TW202140036A - Pharmaceutical compositions of a therapeutic polyene macrolide and methods of their use - Google Patents

Abstract

Disclosed are pharmaceutical compositions including a plurality of nanoparticles including a compound of the following structure:

Description

Pharmaceutical composition of therapeutic polyene macrolide (POLYENE 　 MACROLIDE) and its use method

The present invention relates to pharmaceutical compositions and methods of use.

Macrolide antibiotics include a macrolide ring bonded to one or more deoxysugars. The medical applications of macrolide antibiotics are often limited by their limited storage period and difficulty in achieving effective delivery.

。The compound with the following structure is a therapeutic polyene macrolide:

.

There is a need for new formulations including compound 1 or a pharmaceutically acceptable salt thereof.

， 或其醫藥學上可接受之鹽。In one aspect, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising or including an active pharmaceutical ingredient, and the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt.

， 或其醫藥學上可接受之鹽。In some embodiments, the active pharmaceutical ingredient is

, Or its pharmaceutically acceptable salt.

In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable polymer excipient. In some embodiments, the plurality of nanoparticles comprise pharmaceutically acceptable polymer excipients. In some embodiments, the active pharmaceutical ingredient is nano-encapsulated. In some embodiments, the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate) or polyphosphazene. In some embodiments, the pharmaceutically acceptable polymeric excipient is poly(alkyl cyanoacrylate). In some embodiments, the pharmaceutically acceptable polymer excipients are poly(ethylhexyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(n-hexyl cyanoacrylate), poly(cyanoacrylate) 4-methylpentyl cyanoacrylate), poly(ethyl butyl cyanoacrylate), poly(butyl cyanoacrylate) or poly(octyl cyanoacrylate). In some embodiments, the pharmaceutically acceptable polymeric excipient is poly(ethylhexyl cyanoacrylate). In some embodiments, the pharmaceutically acceptable polymeric excipient is poly(lactic-co-glycolic acid). In some embodiments, the pharmaceutically acceptable polymer excipient is protein (e.g., casein, albumin (e.g., human serum albumin, bovine serum albumin, or ovalbumin), silk protein, gelatin, or a combination thereof) . In some embodiments, the weight ratio of protein to active pharmaceutical ingredient is 1:1 to 20:1 (e.g., 5:1 to 20:1, 1:1 to 5:1, 5:1 to 10:1, or 10: 1 to 20:1).

， 或其醫藥學上可接受之鹽。In some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising poly(ethylhexyl cyanoacrylate) and an active pharmaceutical ingredient, the active pharmaceutical ingredient having the following structure The compound:

, Or its pharmaceutically acceptable salt.

， 或其醫藥學上可接受之鹽。In some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising poly(lactic-co-glycolic acid) and an active pharmaceutical ingredient, the active pharmaceutical ingredient having the following structure Compound:

, Or its pharmaceutically acceptable salt.

， 或其醫藥學上可接受之鹽。In some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising casein, albumin, silk protein, gelatin or a combination thereof and an active pharmaceutical ingredient, the active pharmaceutical ingredient is Compounds with the following structure:

, Or its pharmaceutically acceptable salt.

In some embodiments, the pharmaceutically acceptable polymer excipient is albumin (e.g., human serum albumin, bovine serum albumin, or ovalbumin). In some embodiments, the pharmaceutically acceptable polymer excipient is silk protein. In some embodiments, the pharmaceutically acceptable polymer excipient is gelatin. In some embodiments, the pharmaceutically acceptable polymeric excipient is casein.

In some embodiments, the pharmaceutical composition is a lyophilized composition. In some embodiments, the pharmaceutical composition further includes a plurality of microbubbles.

， 或其醫藥學上可接受之鹽。In another aspect, the present invention provides a pharmaceutical composition comprising a plurality of microbubbles and a plurality of nanoparticles, and the plurality of nanoparticles includes a compound having the following structure:

, Or its pharmaceutically acceptable salt.

， 或其醫藥學上可接受之鹽。In some embodiments, the compound is

, Or its pharmaceutically acceptable salt.

In some embodiments, the nanoparticle comprises poly(alkyl cyanoacrylate), polyphosphazene, or poly(lactic-co-glycolic acid).

In some embodiments, the microbubbles comprise perfluorocarbons, hydrocarbons, sulfur fluoride gas, air, air components, or mixtures thereof. In some embodiments, the microbubbles include nitrogen (N ₂ ), oxygen (O ₂ ), argon (Ar), carbon dioxide (CO ₂ ), helium (He), neon (Ne), methane (CH ₄ ) Or a mixture thereof. In some embodiments, the microbubbles comprise perfluorocarbon. In some embodiments, the microbubbles contain air or its components. In some embodiments, at least a portion of the plurality of nanoparticles are associated with the surface of the microbubbles (for example, the pharmaceutical composition is Pickering emulsion).

In some embodiments, the pharmaceutical composition further comprises a surface active protein (e.g., albumin (e.g., human serum albumin or bovine serum albumin)). In some embodiments, the pharmaceutical composition comprises 0.1% to 2% (e.g., 0.4% to 0.6%, such as 0.5%) (w/w) surface active protein (e.g., albumin (e.g., human serum albumin or bovine serum albumin) )).

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is a nonionic surfactant. In some embodiments, the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitol ester, polysorbate ester, polyethoxylated castor oil, polyoxyethylene /Polyoxypropylene block copolymer or a combination thereof. In some embodiments, the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, or a combination thereof. In some embodiments, the polyoxyethylene fatty acid ester is polyoxyethylated 12-hydroxystearic acid. In some embodiments, the polyoxyethylene ether is polyoxyethylene lauryl ether.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable stabilizer. In some embodiments, the pharmaceutically acceptable stabilizer is vanilla extract, butylated hydroxytoluene, butylated hydroxymethoxybenzene, or vitamin E. In some embodiments, the pharmaceutically acceptable stabilizer is vanilla extract. In some embodiments, relative to the mass of the particles, the pharmaceutical composition contains 0.1%-10% (preferably 0.5%-8% or more preferably 1%-5%) (w/w) pharmaceutically acceptable stabilizer.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable oil. In some embodiments, the pharmaceutically acceptable oil is selected from the group consisting of medium chain triglycerides, long chain triglycerides, and combinations thereof. In some embodiments, the pharmaceutically acceptable oil is one or more medium chain triglycerides. In some embodiments, the one or more medium chain triglycerides are selected from the group consisting of Miglyol, Captex, and Kollisolv. In some embodiments, the pharmaceutical composition contains 0.5%-5% (w/w) pharmaceutically acceptable oil relative to the mass of the particles.

In some embodiments, as measured by dynamic light scattering, the plurality of nanoparticles have an average number average diameter of 20-200 nm (preferably 40-100 nm). In some embodiments, as measured by nanoparticle tracking analysis, the plurality of nanoparticles have an average number average diameter of 30-150 nm (preferably 80-100 nm). In some embodiments, such as in a pharmaceutical composition formulated for oral administration, the polydispersity index of the plurality of nanoparticles is 0.5 or less (e.g., 0.3 or less). In some embodiments, for example, in a pharmaceutical composition formulated for parenteral (e.g., intravenous) administration, the polydispersity index of the plurality of nanoparticles is 0.3 or less (e.g., 0.2 or less) .

In some embodiments, the pharmaceutical composition is an aqueous composition. In some embodiments, the pH of the pharmaceutical composition is 4.0 to 8.0 (for example, the pH is 5.0 to 7.0).

In some embodiments, the pharmaceutical composition includes a co-solvent (e.g., a polar organic solvent). In some embodiments, the polar organic solvent is dimethyl sulfoxide, N-methyl-2-pyrrolidone, N,N-dimethylformamide, or a combination thereof.

In certain preferred embodiments, the pharmaceutical composition is an aqueous composition comprising N-methylpyrrolidone and a pharmaceutically acceptable polymer excipient as poly(lactic-co-glycolic acid).

In certain preferred embodiments, the pharmaceutical composition is an aqueous composition comprising poly(ethylhexyl cyanoacrylate), vanilla extract, and 6-O-palmitoyl-L-ascorbic acid.

In some embodiments, the pharmaceutical composition is an aqueous composition comprising: poly(ethylhexyl cyanoacrylate); vanilla extract; 6-O-palmitoyl-L-ascorbic acid; polyoxyethylated 12- Hydroxystearic acid (e.g. Kolliphor HS 15); polyoxyethylene lauryl ether (e.g. Brij L23); and medium chain triglycerides (e.g. Miglitol).

In some embodiments, the pharmaceutical composition is an aqueous composition comprising poly(lactic-co-glycolic acid), N-methyl-2-pyrrolidone, and polysorbate.

In some embodiments, the pharmaceutical composition is an aqueous composition comprising poly(lactic-co-glycolic acid), N,N-dimethylformamide, and polyoxyethylene/polyoxypropylene block copolymer.

In some embodiments, as measured by liquid chromatography, the pharmaceutical composition contains 1%-15% (e.g. 2%-15%; preferably 3%-10%; more preferably 3.5%-10%; Or better still 3%-6%) (dry weight/dry weight) active pharmaceutical ingredients. In some embodiments, the pharmaceutical composition contains 4.5% to 5.5% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. In some embodiments, the pharmaceutical composition contains 1.5% to 5.5% (eg, 1.5% to 3.0%) (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. In some embodiments, the pharmaceutical composition contains 5% to 10% (dry weight/dry weight) of active pharmaceutical ingredients as measured by liquid chromatography.

In another aspect, the present invention provides a method of treating an individual in need, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition described herein. In another aspect, the present invention provides the use of a plurality of nanoparticles described herein or a pharmaceutical composition described herein for the manufacture of a medicament for treating individuals in need. In another aspect, the present invention provides the pharmaceutical composition described herein for use in the treatment of an individual in need.

In some embodiments, the subject having an infection caused by fungi of the following: Candida (Candida), Cryptococcus (Cryptococcus) genus aspergillus (Aspergillus) genus Colletotrichum anthrax bacteria (Colletotrichum), genus rhusiopathiae (Geotrichum) genus, black yeast-like fungi (Hormonema) genus, oil bottle mold (Lecythophora) genus Paecilomyces (Paecilomyces) genus Penicillium (Penicillium) genus Rhodotorula (Rhodotorula) genus Fusarium (of Fusarium) genus bacteria, yeast (Saccharomyces), Trichoderma (Trichoderma) genus Trichophyton (Trichophyton) is a small broom-like mold (Scopularilopsis) species, Histoplasma (Histoplasma) genus, blastomycosis (Blastomyces) genus or coccidioidomycosis ( Cocciodioides ) genus. In some embodiments, the individual has a fungal infection caused by: Candida, Aspergillus, or Cryptococcus. In some embodiments, the individual has a fungal infection caused by azole-resistant Aspergillus sp.

In some embodiments, the pharmaceutical composition is intravenous, by inhalation, intranasal, oral, sublingual, buccal, transdermal, intradermal, intramuscular, intravaginal, parenteral, intraarterial, intracranial , Intrathecal, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal or local administration.

In yet another aspect, the present invention provides a method of delivering a therapeutically effective amount of Compound 1 , Compound 1A, or a pharmaceutically acceptable salt thereof to a target site in an individual, the method comprising administering to the individual the medicine described herein combination. In yet another aspect, the present invention provides the pharmaceutical composition described herein for the manufacture of a medicament for delivering a therapeutically effective amount of Compound 1 , Compound 1A, or a pharmaceutically acceptable salt thereof to a target site in an individual use. In yet another aspect, the present invention provides the pharmaceutical composition described herein for delivering a therapeutically effective amount of Compound 1 , Compound 1A, or a pharmaceutically acceptable salt thereof to a target site in an individual.

In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the target site is the individual's lungs.

， 或其醫藥學上可接受之鹽； 該方法包含使於一液體中之該醫藥學上可接受之聚合物賦形劑之單體前驅體與該化合物或其醫藥學上可接受之鹽聚合，該液體包含該單體前驅體，其中該聚合步驟產生該複數個奈米粒子。In yet another aspect, the present invention provides a method for generating a plurality of nanoparticles, the plurality of nanoparticles comprising a pharmaceutically acceptable polymer excipient and a compound having the following structure:

, Or a pharmaceutically acceptable salt thereof; the method comprises polymerizing a monomer precursor of the pharmaceutically acceptable polymer excipient and the compound or a pharmaceutically acceptable salt thereof in a liquid , The liquid contains the monomer precursor, and the polymerization step produces the plurality of nanoparticles.

， 或其醫藥學上可接受之鹽。In some embodiments, the compound has the following structure:

, Or its pharmaceutically acceptable salt.

In some embodiments, the liquid further includes a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is a nonionic surfactant.

In some embodiments, the liquid further includes a pharmaceutically acceptable stabilizer. In some embodiments, the liquid further comprises a pharmaceutically acceptable oil.

In some embodiments, the monomer precursor is alkyl cyanoacrylate, and the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate).

In some embodiments, as measured by dynamic light scattering, the plurality of nanoparticles have an average number average diameter of 20-200 nm (preferably 40-100 nm). In some embodiments, as measured by Nanoparticle Tracking Analysis (NTA), the plurality of nanoparticles have an average number average diameter of 30-150 nm (preferably 80-100 nm).

In some embodiments, the liquid is an aqueous composition. In some embodiments, the pH of the liquid is 0.5 to 8.0 (for example, the pH is 0.5 to 3.0). In some embodiments, the pH of the liquid is 2.0 to 8.0 (preferably, the pH is 3.0 to 7.0).

In some embodiments, the method further includes adding a plurality of microbubbles. In some embodiments, the method further includes freeze-drying the plurality of nanoparticles. In some embodiments, the method further comprises dialyzing the plurality of nanoparticles with deionized water. In some embodiments, the method further comprises adjusting the pH of the liquid to be in the range of 4.0 to 8.0 (preferably in the range of 5.0 to 7.0).

In some embodiments, the step of adjusting the pH is performed during the polymerization step. definition

The term "about" as used herein means a value within ±10% of the value following the term "about".

The term "(dry weight/dry weight)" percentage as used herein refers to the weight percentage of ingredients in a composition that does not include a liquid pharmaceutically acceptable carrier. The (dry weight/dry weight) percentage can be measured using, for example, liquid chromatography.

The term "nanoparticle" as used herein refers to a population of particles having a Z-average diameter of less than 1000 nm as measured by dynamic light scattering.

The term "pharmaceutical composition" as used herein refers to a composition formulated with pharmaceutically acceptable excipients and used as part of a therapeutic regimen for treating diseases in mammals.

The term "pharmaceutical dosage form" as used herein refers to those medicines that are intended to be administered to an individual without further modification (e.g., not diluted with a liquid solvent, not suspended in a liquid solvent, or not dissolved in a liquid solvent). combination.

The term "pharmaceutically acceptable excipient" as used herein refers to any ingredient other than one or more active agents described herein (e.g., a vehicle capable of suspending or dissolving one or more active agents) and It has the characteristics of being substantially non-toxic and substantially non-inflammatory in patients. Excipients may include, for example, antioxidants, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), flavoring agents, fragrances, preservatives, printing inks, adsorbents, suspending agents Or dispersant, sweetener, liquid solvent and buffer.

As used herein, the term "pharmaceutically acceptable salt" refers to those that are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without abnormal toxicity, irritation, allergic reactions, and the like, and are reasonable The benefit/risk ratio is commensurate with their salts. Pharmaceutically acceptable salts are known in the art. For example, pharmaceutically acceptable salts are described in the following: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use , (editors PH Stahl and CG Wermuth) , Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting free base groups with suitable organic acids. Examples of suitable acids for forming such salts include acetic acid, aspartic acid, benzenesulfonic acid, benzoic acid, dicarbonic acid, disulfuric acid, ditartaric acid, butyric acid, calcium edetate, camphorsulfonic acid, carbonic acid, chlorine Benzoic acid, citric acid, edetic acid, ethanedisulfonic acid, dodecylsulfonic acid (estolic), esyl, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid , Glutamic acid, hydantoin phenylarsine acid, cyclohexylamine sulfonic acid, hexyl isophthalic acid, hypamine acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, Lactic acid, lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, methyl nitric acid, methyl sulfuric acid, mucic acid, muconic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, Nitromethanesulfonic acid, phosphatic acid, pantothenic acid, phosphoric acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, phthalic acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, succinic acid, aminosulfonic acid , P-aminobenzenesulfonic acid, sulfonic acid, sulfuric acid, tannic acid, tartaric acid, theochloric acid (teoclic) and toluenesulfonic acid. Glutamate is especially preferred.

The term "individual" as used herein means as determined by a qualified professional (such as a doctor or nurse physician) with or without one or more laboratory tests from one or more samples from an individual known in the art, A human or non-human animal (e.g., a mammal) suffering from a disease, disorder, or condition or at risk of the disease, disorder, or condition. Non-limiting examples of diseases, disorders, and conditions include fungal infections caused by: Candida, Cryptococcus, Aspergillus, Colletotrichum anthracis, Geotrichum, Black yeast-like fungi, Oil bottle Molds, Paecilomyces, Penicillium, Rhodotorula, Fusarium, Saccharomyces, Trichoderma, Trichophyton, and Sclerotium. Preferably, the fungal infection is caused by Candida, Aspergillus or Cryptococcus. More preferably, the fungal infection is caused by azole-resistant Aspergillus sp.

"Treatment/treating" as used herein refers to the medical management of an individual who intends to improve, alleviate, stabilize, prevent, or cure a disease, disorder, or condition. This term includes active treatment (treatment for improving the disease, disease, or condition); etiological treatment (treatment for the cause of the related disease, disease, or condition); palliative treatment (designed to relieve the symptoms of a disease, disease, or condition) Treatment); preventive treatment (treatment aimed at minimizing or partially or completely inhibiting the development of related diseases, disorders, or conditions); and supportive treatment (treatment to supplement another therapy).

， 或其醫藥學上可接受之鹽。Generally speaking, the present invention provides a pharmaceutical composition including a plurality of nanoparticles and a method of using the same. The pharmaceutical composition of the present invention includes a plurality of nanoparticles, the plurality of nanoparticles includes an active pharmaceutical ingredient, and the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt.

， 或其醫藥學上可接受之鹽。In some embodiments, the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt.

The nanoparticles described herein may include polymer excipients (e.g., polymer nanoparticles) or may include lipids (e.g., lipid nanoparticles, such as liposomes, micelles, etc.).

The nanoparticles described herein may include lipids, such as phospholipids (e.g., phospholipid choline, phosphatidic acid, phospholipid serine, phospholipid ethanolamine, or phospholipid glycerol). In some embodiments, the nanoparticles described herein include phospholipid choline (e.g. dipalmitoyl phospholipid choline, distearyl phospholipid choline, lecithin phospholipid choline, and soy phospholipid choline). Alkali) or phospholipids (for example dipalmitoyl phospholipid glycerol, distearyl phospholipid glycerol, dilauryl phospholipid glycerol or dimyristyl phospholipid glycerol). For example, lipids (such as phospholipids) can encapsulate active pharmaceutical ingredients in vesicles or micelles.

Preferably, the pharmaceutical composition described herein includes a pharmaceutically acceptable polymer excipient (e.g. poly(alkyl cyanoacrylate), poly(lactic acid-co-glycolic acid)) or protein (e.g. albumin, Silk protein, gelatin, casein or a combination thereof)). Advantageously, the pharmaceutical compositions described herein can exhibit a commercially acceptable shelf life. Without wishing to be bound by theory , the encapsulation of compound 1 (such as compound 1A ) or its pharmaceutically acceptable salt in a pharmaceutically acceptable polymer excipient can be compound 1 (such as compound 1A). ) Or its pharmaceutically acceptable salt provides sufficient stability to have a commercially acceptable shelf life. For example, the pharmaceutical composition described herein can retain 90% to 110% of the label dose of Compound 1 (such as Compound 1A ) or a pharmaceutically acceptable salt thereof after being stored at 4°C for two weeks.

As measured by liquid chromatography, the pharmaceutical composition described herein may contain at least 2%, preferably at least 3%, and in particular at least 5% (dry weight/dry weight) of compound 1 or its pharmacologically An acceptable salt (for example, Compound 1A or a pharmaceutically acceptable salt thereof). As measured by liquid chromatography, the pharmaceutical composition described herein may contain at least 2% (e.g., at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, or at least 9.5%) (dry weight/dry weight) compound 1 or its pharmaceutically acceptable Accepted salt (e.g. Compound 1A or a pharmaceutically acceptable salt thereof). As measured by liquid chromatography, the pharmaceutical composition described herein may contain up to 15%, preferably up to 12%, and specifically up to 10% (dry weight/dry weight) of compound 1 or its pharmacologically An acceptable salt (for example, Compound 1A or a pharmaceutically acceptable salt thereof). As measured by liquid chromatography, non-limiting examples of the range include 2%-15%, preferably 3%-10% and specifically 3%-6% (e.g. 3.5%-15%, 4% -15%, 4.5%-15%, 5%-15%, 5.5%-15%, 6%-15%, 6.5%-15%, 7%-15%, 7.5%-15%, 8%-15 %, 8.5%-15%, 9%-15%, 9.5%-15%, 3%-10%, 3.5%-10%, 4%-10%, 4.5%-10%, 5%-10%, 5.5%-10%, 6%-10%, 6.5%-10%, 7%-10%, 7.5%-10%, 8%-10%, 8.5%-10%, 9%-10%, 9.5% -10%, 3%-7.5%, 3.5%-7.5%, 4%-7.5%, 4.5%-7.5%, 5%-7.5%, 5.5%-7.5%, 6%-7.5%, 6.5%-7.5 %, 7%-7.5%, 3%-5%, 3.5%-5%, 4%-5% or 4.5%-5%) (dry weight/dry weight) compound 1 or its pharmaceutically acceptable salt (For example, compound 1A or a pharmaceutically acceptable salt thereof).

The pharmaceutical composition described herein contains a plurality of nanoparticles. As measured by dynamic light scattering, the plurality of nanoparticles may have an average number average diameter of, for example, 20-200 nm (preferably, the average number average diameter is 40-100 nm). Preferably, as measured by nanoparticle tracking analysis (NTA), the plurality of nanoparticles have an average number average diameter of 30-150 nm (more preferably, the average number average diameter is 80-100 nm).

The pharmaceutical composition described herein includes one or more pharmaceutically acceptable excipients (e.g., pharmaceutically acceptable polymer excipients), surfactants (e.g., nonionic surfactants), stabilizers, Carriers (e.g. oils) and/or flavoring agents.

In the pharmaceutical compositions described herein, polymeric excipients can be used, for example, to encapsulate active pharmaceutical ingredients. Non-limiting examples of pharmaceutically acceptable polymeric excipients include poly(alkyl cyanoacrylate), poly(lactic-co-glycolic acid), amphiphilic polyphosphazene, and protein. Preferably, the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate) (e.g. poly(ethylhexyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(cyanoacrylate) N-hexyl acrylate), poly(4-methylpentyl cyanoacrylate), poly(ethylbutyl cyanoacrylate), poly(butyl cyanoacrylate) or poly(octyl cyanoacrylate)). More preferably, the pharmaceutically acceptable polymer excipient is poly(ethylhexyl cyanoacrylate) (for example, poly(2-ethylhexyl cyanoacrylate)). Preferred proteins include albumin, silk protein, gelatin, casein and combinations thereof.

Nanoparticles including poly(alkyl cyanoacrylate) can be prepared in situ by polymerizing alkyl cyanoacrylate monomers in a composition including compound 1 , 1A or a pharmaceutically acceptable salt thereof. The method of preparing poly(alkyl cyanoacrylate) nanoparticles can include, for example, customizing the specific surface of the nanometer by introducing hydrophilic polymers, such as pharmaceutically acceptable polymer excipients. Agents and, for example, surfactants having reactive moieties capable of reacting with pharmaceutically acceptable polymer excipient precursors (e.g., monomers that produce pharmaceutically acceptable polymer excipients). The polymerization of monomers can be initiated using such surfactants. Alternatively, the polymerization of monomers can be initiated using a polymerization initiator, such as an azo initiator (for example, 2,2'-azobis(isobutyronitrile), dimethyl 2,2'- Azobis(2-methylpropionate), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2- Methylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile) or 2,2'-azobis(N-butyl-2-methylpropionamide)). In some embodiments, the pharmaceutical composition described herein includes nanoparticles, the nanoparticles include pharmaceutically acceptable polymer excipients and as compound 1 , 1A or a pharmaceutically acceptable salt thereof Active pharmaceutical ingredients. Preferably, the nanoparticles are coated with polyethylene glycol (PEG). Advantageously, PEG coated on nanoparticles can reduce clearance by, for example, the immune system.

Surfactants can be used to stabilize the pharmaceutical composition against, for example, crystallization and mechanical stress (such as agitation and/or shear). Surfactants can be nonionic or ionic. Non-limiting examples of nonionic surfactants include polyoxyethylene ethers, polyoxyethylene esters (e.g. polyoxyethylene fatty acid esters), sorbitol esters, polysorbate esters, sorbitol, ethoxylated phenols , Ethoxylated diphenol, polyethoxylated castor oil, polyoxyethylene/polyoxypropylene block copolymer (e.g. poloxamer), poloxamine, fatty acid monoglyceride , Fatty acid diglycerides, polysaccharides (such as hyaluronic acid or sialic acid), proteins (such as albumin or casein), and combinations thereof. Preferably, the surfactant is polyoxyethylene ether or polysorbate. More preferably, the surfactant is polyoxyethylated 12-hydroxystearic acid (for example, Cleaver HS 15), polyoxyethylene lauryl ether (for example, Bridger L23), or a combination thereof. Non-limiting examples of ionic surfactants include, for example, sodium lauryl sulfate, sodium lauryl sulfate, sulfosuccinates, and fatty acid salts.

The surfactant can be covalently attached to the polymeric excipient. In a non-limiting example, the surfactant described herein can be included in a monomer mixture of solvents, active agents, and polymer excipients. Therefore, the surfactant (such as the free -OH group in the surfactant) can initiate the polymerization of the monomer (such as alkyl cyanoacrylate) to encapsulate the active agent in the surfactant-coated nanoparticle ( For example, PEG-coated nanoparticles).

Stabilizers can be used to stabilize pharmaceutical compositions against, for example, oxidative stress. Non-limiting examples of stabilizers include vanilla extract, butylated hydroxytoluene, butylated hydroxymethoxybenzene, vitamin E, and 6-O-palmitoyl-L-ascorbic acid. Preferably, the stabilizer is vanilla extract. Relative to the particle mass, the pharmaceutical composition may include, for example, 0.1%-10%, preferably 0.5%-8%, and specifically 1%-5% (e.g., 0.1%-9%, 0.1%-8%, 0.1% %-7%, 0.1%-6%, 0.1%-5%, 0.1%-4%, 0.1%-3%, 0.1%-2%, 0.1%-1%, 0.1%-0.5%, 0.2%- 10%, 0.2%-9%, 0.2%-8%, 0.2%-7%, 0.2%-6%, 0.2%-5%, 0.2%-4%, 0.2%-3%, 0.2%-2% , 0.2%-1%, 0.2%-0.5%, 0.5%-10%, 0.5%-9%, 0.5%-8%, 0.5%-7%, 0.5%-6%, 0.5%-5%, 0.5 %-4%, 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%- 6%, 1%-5%, 1%-4%, 1%-3%, 1%-2%, 2%-10%, 2%-9%, 2%-8%, 2%-7% , 2%-6%, 2%-5%, 2%-4%, 2%-3%, 3%-10%, 3%-9%, 3%-8%, 3%-7%, 3 %-6%, 3%-5%, 3%-4%, 4%-10%, 4%-9%, 4%-8%, 4%-7%, 4%-6%, 4%- 5%, 5%-10%, 5%-9%, 5%-8%, 5%-7%, 5%-6% or 5%) (w/w) stabilizer.

The carrier can be used to suspend or dissolve the active pharmaceutical ingredient in the pharmaceutical composition. The carrier can also be used to prevent Ostwald ripening during the preparation of the formulation. The suspending or dissolving vehicle may be an aqueous vehicle, such as water or saline (e.g., isotonic saline). In addition, non-limiting examples of pharmaceutically acceptable carriers include pharmaceutically acceptable oils, such as medium chain triglycerides, long chain triglycerides, or combinations thereof. Preferably, the pharmaceutically acceptable oil is one or more medium-chain triglycerides (for example, Miglitol, Carport, and Klisov). Relative to the particle mass, the pharmaceutical composition may include, for example, 0.5-5%, preferably 1-5%, and specifically 2-3% (e.g., 0.5%-5%, 0.5%-4.5%, 0.5%-4 %, 0.5%-3.5%, 0.5%-3%, 0.5%-2.5%, 0.5%-2%, 0.5%-1.5%, 0.5%-1%, 1%-5%, 1%-4.5%, 1%-4%, 1%-3.5%, 1%-3%, 1%-2.5%, 1%-2%, 1%-1.5%, 1.5%-5%, 1.5%-4.5%, 1.5% -4%, 1.5%-3.5%, 1.5%-3%, 1.5%-2.5%, 1.5%-2%, 2%-5%, 2%-4.5%, 2%-4%, 2%-3.5 %, 2%-3%, 2%-2.5%, 2.5%-5%, 2.5%-4.5%, 2.5%-4%, 2.5%-3.5%, 2.5%-3%, 3%-5%, 3%-4.5%, 3%-4%, 3%-3.5%, 3.5%-5%, 3.5%-4.5%, 3.5%-4%, 4%-5%, 4%-4.5% or 4.5% -5%) (w/w) Medically acceptable oil.

For oral administration of the pharmaceutical composition, flavoring agents may be included to make it more palatable. Any effective flavoring agent can be used. The flavoring agent can be a natural flavoring agent, an artificial flavoring agent, or a mixture thereof. The flavoring agent produces a flavor that will help reduce the undesired taste of the active ingredient. In one embodiment, the flavoring agent can produce mint, menthol, honey lemon, orange, lemon lime, grape, cranberry, vanilla berry, bubble gum, or cherry flavor. Flavoring agents can be natural or artificial sweeteners, such as sucrose, ammonium glycyrrhizinate (Magnasweet), sucralose, xylitol, sodium saccharin, cyclamate, aspartame, acesulfame Acid and its salts.

The pharmaceutical composition described herein may be an aqueous composition (e.g., a suspension). The pH of the pharmaceutical composition can be, for example, 4.0 to 8.0 (preferably 5.0 to 7.0). Alternatively, the pharmaceutical composition may be a lyophilized composition. The lyophilized composition can be reconstituted before use to produce an aqueous composition.

The pharmaceutical compositions described herein can be used to treat individuals in need. The method of treating an individual includes administering to the individual a therapeutically effective amount of the pharmaceutical composition described herein. The individual may suffer from fungal infections (such as invasive fungal infections) caused by, for example, Candida, Cryptococcus, Aspergillus, Colletotrichum anthracis, Geotrichum, Black yeast-like fungi, oil Phialomyces, Paecilomyces, Penicillium, Rhodotorula, Fusarium, Saccharomyces, Trichoderma, Trichophyton, Sclerotium, Histoplasma, Blastomyces, or Coccidioides Bacteria. Except for one or more of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, chronic pulmonary aspergillosis, recovery from solid organ transplantation, recovery from hematology transplantation, and immunosuppression after cancer chemotherapy, Individuals may also have fungal infections (e.g., invasive fungal infections). Preferably, the fungal infection is caused by Candida, Aspergillus or Cryptococcus. More preferably, the fungal infection is caused by azole-resistant Aspergillus sp.

The pharmaceutical compositions described herein can be administered to an individual in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from each other by, for example, 1-24 hours, 1-7 days, 1-4 weeks, or 1-12 months. The pharmaceutical composition may be administered according to a schedule, or the pharmaceutical composition may not be administered according to a predetermined schedule. It should be understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the pharmaceutical composition or supervising the administration of the pharmaceutical composition.

Although the attending physician finally determines the appropriate amount and dosage regimen, for example, the effective amount of the compound of the present invention can be any of the active pharmaceutical ingredients (API) described herein, for example, between 0.05 mg and 3000 mg The total daily dose. Alternatively, the dosage can be calculated using the weight of the patient.

In the method of the present invention, the time period during the administration of multiple doses of the pharmaceutical composition to the individual is variable. In some embodiments, a certain dose of the pharmaceutical composition is administered to the individual over a period of 1-7 days, 1-12 weeks, or 1-3 months. In some embodiments, the pharmaceutical composition is administered to the individual over a period of, for example, 4-11 months or 1-30 years. In some embodiments, the pharmaceutical composition is administered to the individual at the onset of symptoms. In any of these embodiments, the amount of the pharmaceutical composition administered can vary during the administration period. When the pharmaceutical composition is administered daily, the administration can occur, for example, 1-12 times a day.

Exemplary administration routes of the pharmaceutical compositions described herein include intravenous, inhalation, intranasal, oral, sublingual, buccal, transdermal, intradermal, intramuscular, intravaginal, parenteral, intraarterial, Intracranial, intrathecal, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal and local administration. Preferably, the route of administration is intravenous.

The pharmaceutical composition described herein includes a pharmaceutical composition formulated for intravenous or intraarterial administration. The pharmaceutical composition described herein may include the microbubbles and nanoparticles described herein. Preferably, the pharmaceutical composition including microbubbles and nanoparticles is formulated for intravenous administration. Advantageously, a pharmaceutical composition including the microbubbles and nanoparticles described herein can help target the delivery of Compound 1 or 1A to target tissues (e.g., lung and/or heart).

The pharmaceutical composition may include microbubbles, such as any conventional commercially available contrast microbubbles, such as Albunex (GE Healthcare), Optison (GE Healthcare), Sonazoid (GE Healthcare), Sonovue (Bracco) or other conventional contrast microbubbles known to those skilled in the art. In some pharmaceutical compositions, the surface of the microbubbles can be associated with nanoparticles. The microbubbles can be generated, for example, in a nanoparticle solution as described herein. Advantageously, the nanoparticles can have a stabilizing effect on the surface of the microbubbles. The microbubbles may be, for example, microbubbles filled with gas or its precursors. The gas may include or may be, for example, perfluorocarbon, hydrocarbon (e.g., methane), sulfur fluoride (e.g., SF ₆ ), halogen, air, air components (e.g., nitrogen (N ₂ ), oxygen (O ₂ ), argon ( Ar), carbon dioxide (CO ₂ ), helium (He) or neon (Ne)) or mixtures thereof. Preferably, the gas is perfluorocarbon, air, air components (for example, nitrogen (N ₂ ), oxygen (O ₂ ), argon (Ar), carbon dioxide (CO ₂ ), helium (He) or neon ( Ne)) or a mixture thereof. More preferably, the gas is perfluorocarbon.

Without wishing to be bound by theory, the solubility of gas in microbubbles can affect the ability of microbubbles to circulate in the blood and accumulate in the respiratory system. For example, microbubbles filled with perfluorocarbon gas can have an extended cycle time. Without wishing to be bound by theory, the extended circulation time can be attributed to the low solubility of perfluorocarbons in the blood. The pharmaceutical compositions described herein may include microbubbles including gases such as perfluorocarbons. Alternatively, the gas may be, for example, air or a component thereof. Alternatively, the gas may be, for example, sulfur fluoride gas, preferably sulfur hexafluoride (SF ₆ ) gas.

Commercially available microbubbles are usually provided in the form of a gaseous microbubble suspension stabilized by a shell of lipids, proteins and/or other surfactants. Microbubbles can be made, for example, with surface active compounds such as proteins, polymers, lipids, surfactants or mixtures thereof. One or more surface active compounds can stabilize the microbubbles. Preferred non-limiting examples of surface active proteins are albumin (such as human or bovine serum albumin or from other suitable biocompatible albumin sources, including synthetic albumin) and casein. A preferred non-limiting example of surface active lipids is phospholipids. Microbubbles may also contain, for example, additional stabilizers and excipients, such as cholesterol or polyoxyethylene-polyoxypropylene.

The pharmaceutical compositions described herein may include, for example, modifiers. Modifiers can modify the interaction between the components of the pharmaceutical composition. The modifier can form complexes or cross-links between the microbubbles and/or surface active compounds and the nanoparticles, for example, to improve the stability of the pharmaceutical composition. The modifier can, for example, introduce an interaction between the surface active compound and the nanoparticle. The modifier may be, for example, urea (H ₂ N-CO-NH ₂ ). Preferably, the pharmaceutical composition includes protein (preferably casein) as a surface active compound and urea as a modifier. Urea can act as a denaturant for proteins. Without wishing to be bound by theory, it is believed that urea may interfere with the hydrogen bonds involved in protein folding. Urea can also form a complex with acid groups on the surface of the nanoparticle and improve the hydrophilicity of the nanoparticle.

In pharmaceutical compositions that include surface active compounds (such as proteins) and urea, the amount of active agent delivered to the target tissue is increased. By introducing a strong interaction between the surface active compound and the nanoparticle, urea can stabilize the microbubbles. Without being bound by theory, it is believed that modified microbubbles, surface active compounds and/or nanoparticles can enhance the stability of the association between microbubbles and nanoparticles in pharmaceutical compositions. As the association between microbubbles and nanoparticles increases, the number of nanoparticles delivered to the target lung tissue can be greater than the number of nanoparticles in a composition lacking an agent that enhances the association between microbubbles and nanoparticles .

The pharmaceutical composition described herein including microbubbles and nanoparticles may be Pickering emulsion. Hydrophobic solid particles can be strongly adsorbed at the interface between immiscible fluids such as oil and water, thus forming Pickering emulsion-an emulsion stabilized by solid nanoparticles or microparticles. Therefore, the nanoparticle associated with the surface of the microbubbles can stabilize the composition in the form of a Pickering emulsion. Advantageously, the pharmaceutical compositions described herein can be further stabilized in the form of Pickering emulsions by formulations. The average diameter of the microbubbles associated with the nanoparticle can be, for example, 0.5 μm to 30 μm (for example, 1-10 μm). The diameter of the microbubble can be measured, for example, by two-dimensional analysis of the microbubble image using the ImageJ image analyzer.

In addition, for example, the pharmaceutical composition described herein may also include free nanoparticles in addition to the nanoparticles associated with the surface of the microbubbles. The pharmaceutical composition described herein may include nanoparticles associated with the microbubbles described herein and free nanoparticles described herein.

A pharmaceutical composition including the microbubbles and nanoparticles described herein can be prepared, for example, according to a method including combining the gas or microbubbles described herein with the nanoparticles. For example, the nanoparticles can be in solution. Nanoparticles can be prepared in situ as described herein, or can be reconstituted from a dry composition.

The pharmaceutical composition including the microbubbles and nanoparticles described herein can be prepared, for example, according to a method including the following: a. Add the microbubbles described in this article and the nanoparticles described in this article to the solution, and b. Mix the solution to produce a pharmaceutical composition.

Alternatively, a pharmaceutical composition including the microbubbles and nanoparticles described herein can be prepared, for example, according to a method including the following: a. Synthesize the nano particles described in this article, b. Combination of nano particles and surface active compounds, c. Add gas to the solution, and d. Mix the solution to produce a pharmaceutical composition.

In some embodiments, the solution is mixed (e.g., stirred) for 2 seconds to 60 minutes (e.g., 1-10 minutes). For example, solution mixing methods such as ultrasonic treatment, mechanical stirring, microfluidics, and shaking are known in the art. In some preparation methods for microbubble-containing formulations, the composition can be degassed before adding microbubble gas. Degassing methods are known in the art; non-limiting examples of degassing methods include, for example, sonic processing and freeze-pump-thaw degassing.

The pharmaceutical composition described herein includes a pharmaceutical composition formulated for oral administration ("oral dosage form"). The oral dosage form may, for example, be in the form of tablets, capsules, liquid suspensions, powders, granules or pellets containing active pharmaceutical ingredients and one or more pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers; granulating agents and disintegrating agents; binders; and lubricants, slip agents, anti-sticking agents, coloring agents, flavoring agents, plasticizers, and humectants , Buffer and similar reagents.

Controlled release compositions for oral use can be constructed to release the active drug by controlling the dissolution and/or diffusion of the active pharmaceutical ingredient. Dissolution or diffusion controlled release can be achieved by appropriately coating tablets, capsules, pellets, granules or particles containing API or by incorporating particles containing API into a suitable matrix. In some embodiments, the composition includes a biodegradable, pH and/or temperature sensitive polymer coating. For example, oral dosage forms may include active pharmaceutical ingredients (such as nanoparticles described herein).

The pharmaceutical compositions described herein can be prepared using the techniques and methods described herein and techniques and methods known in the art.

， 或其醫藥學上可接受之鹽。The present invention is further characterized by a method for producing a plurality of nanoparticles, the plurality of nanoparticles includes an active pharmaceutical ingredient, and the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt.

In particular, the plurality of nanoparticles may include, for example, pharmaceutically acceptable polymer excipients.

Therefore, the method includes polymerizing a monomer precursor of a pharmaceutically acceptable polymer excipient and a compound or a pharmaceutically acceptable salt thereof in a liquid that includes the monomer precursor. The polymerization step produces a plurality of nanoparticles.

， 或其醫藥學上可接受之鹽。In some embodiments, the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt.

In the production method described herein, the liquid may further include a pharmaceutically acceptable surfactant (for example, a nonionic surfactant, such as polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitol ester, polysorbate). Sugar alcohol ester, polyethoxylated castor oil, polyoxyethylene/polyoxypropylene block copolymer or a combination thereof). Preferably, the pharmaceutically acceptable surfactant is polyoxyethylene ether (for example, polyoxyethylene lauryl ether), polyoxyethylene fatty acid ester (for example, polyoxyethylated 12-hydroxystearic acid) or a combination thereof .

In the production method described herein, the liquid may further include a pharmaceutically acceptable stabilizer (for example, vanilla extract, butylated hydroxytoluene, butylated hydroxymethoxybenzene, or vitamin E). Preferably, the pharmaceutically acceptable stabilizer is vanilla extract.

In the production method described herein, the liquid may further include a pharmaceutically acceptable oil (for example, an oil selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, and combinations thereof). Preferably, the pharmaceutically acceptable oil is one or more medium-chain triglycerides (for example, Miglitol, Carport, and Klisov).

In the production method described herein, the monomer precursor can be, for example, alkyl cyanoacrylate (e.g., ethylhexyl cyanoacrylate), and the pharmaceutically acceptable polymer excipient can be, for example, poly(cyanoacrylate). Alkyl acrylate) (e.g. poly(ethylhexyl cyanoacrylate)).

In the production method described herein, as measured by dynamic light scattering, a plurality of nanoparticles may have an average number average diameter of 20-200 nm (for example, 40-100 nm). In the production method described herein, as measured by nanoparticle tracking analysis, a plurality of nanoparticles may have an average number average diameter of 30-150 nm (for example, 80-100 nm).

The liquid may be, for example, an aqueous composition (e.g., an aqueous composition having a pH of 0.5 to 8.0 (e.g., a pH of 0.5 to 3.0, 2.0 to 8.0, or 3.0 to 7.0)).

The production method may further include a step of freeze-drying a plurality of nanoparticles. Additionally or alternatively, the production method may further include a step of dialyzing a plurality of nanoparticles with deionized water.

In the production method described in this article, the pH of the liquid can be adjusted as needed. For example, the pH of the liquid can be adjusted to be in the range of 4.0 to 8.0 (e.g., 5.0 to 7.0). In some embodiments, the step of adjusting the pH is performed during the polymerization step. In other embodiments, the step of adjusting the pH is performed before or after the polymerization step.

The following examples are intended to illustrate the invention. These examples are not intended to limit the invention in any way.

Examples Example 1. Preparation of polyphosphazene nanoparticle composition using ethyl 4-aminobenzoate (CAS: 94-09-7) with a molecular weight of 7 kDa to 11 kDa and polyvinyl alcohol substitution (8 to 13 repeats) Unit) amphiphilic polyphosphazene (POPZ) polymer (SINTEF). Dissolve 11 mg of compound 1A (customized) in 11 mL DMF and 100 mg POPZ. The solution was added dropwise to 11 mL of distilled water under strict stirring at room temperature. In the case of one displacement, the sample was dialyzed with distilled water. The particle solution was lyophilized to produce dry nanoparticle powder with a theoretical compound 1A loading amount of 10% (w/w).

Example 2. pH = 2 Preparation of poly (2-ethylhexyl cyanoacrylate) nano-granular composition was prepared containing in 12 mL 0.01 M HCl (pH 2 ) in the PEG stabilizer Cleaver HS 15 (0.12 g) And Bridger L23 (0.12 g) in water. Prepare a solution containing 0.096 g compound 1A (customized) dissolved in 2-ethylhexyl cyanoacrylate (0.8 g) and stabilizer (0.05 g vanilla extract and 15 μL miglitol 812), and store it in the room Keep stirring at temperature for 2 hours.

The two solutions were mixed on ice and homogenized on an ultrasonic processor (50% amplitude) for 3 minutes, with 10 seconds pause every 30 seconds. The nanoemulsion was polymerized on a spinner at room temperature for three hours. The pH was adjusted to pH 6 with 0.1 M NaOH and further polymerized at room temperature overnight (on a rotator). The particle sample was dialyzed with distilled water. The final product is a liquid suspension. The theoretical load is 10% (w/w).

Example 3. Preparation of pH = 4 The same procedure of poly (2-ethylhexyl cyanoacrylate) using the nano granule composition described in Example 2. The procedure for this example, except that an aqueous solution containing 0.1 mM HCl (pH = 4) instead of 0.01 M HCl.

Example 4. Preparation of poly pH = 1 The same procedure (2-ethylhexyl cyanoacrylate) using the nano granule composition described in Example 2. The procedure for this example, except that an aqueous solution containing 0.1 M HCl (pH = 1) instead of 0.01 M HCl.

Example 5. In the case of non-L23 Bridger same procedure for the preparation of poly (2-ethylhexyl cyanoacrylate) nano-granular composition in Example 2-4 using the procedure described for this example, except The aqueous solution contained does not contain Bridger L23 and the amount of Clifford HS 15 is doubled.

Example 6. In the case of vanilla extract more of the same procedure for the preparation of poly (2-ethylhexyl cyanoacrylate) nano-granular composition in Example 2-5 using the procedure described for this example, except The reason is that the amount of vanilla extract is increased to 0.1 g.

Example 7. In the case of vanilla extract more of the same procedure for the preparation of poly (2-ethylhexyl cyanoacrylate) nano-granular composition in Example 2-5 using the procedure described for this example, except The point is that the amount of vanilla extract is reduced to 0.025 g.

Example 8. Preparation of pH = 2, the theoretical load = 14.8% of poly (2-ethylhexyl cyanoacrylate) nano particle composition using the procedure of Example 2, the same as described in the procedure for this example, different The point is that the theoretical loading of compound 1A is 14.8% (w/w). The measured final loading of compound 1A was 3.3% (w/w).

Example 9. Preparation of pH = 3, the theoretical load = 14.8% of poly (2-ethylhexyl cyanoacrylate) nano particle composition using the procedure of Example 2, the same as described in the procedure for this example, different The point is that (1) the aqueous solution contains 1 mM HCl (pH = 3) instead of 0.01 M HCl, and (2) the theoretical loading of compound 1A is 14.8% (w/w). The measured final loading of compound 1A was 3.6% (w/w).

Example 10. Preparation of pH = 4, the theoretical load = 14.8% of poly (2-ethylhexyl cyanoacrylate) using the nano granule composition described in Example 3. The procedure of the same procedure used in this example, different The point is that the theoretical loading of compound 1A is 14.8% (w/w). The measured final loading of compound 1A was 3.8% (w/w).

Example 11. Physicochemical characterization of nanoparticle composition. Dynamic light scattering (Malvern Zetasizer and Nanoparticle Tracking Analyzer (NTA)) was used to measure size and size distribution in a phosphate buffer (pH 7). The dry weight was determined by drying three sample aliquots at 50°C overnight. Weigh/pipette three aliquots of samples, dissolve them in DMSO and dilute to determine drug loading and stability for LC-DAD-QTOF analysis. Three samples of Amphotericin B USP were used as standards to determine the concentration of compound 1A. The LC-QTOF method is: Mobile phase: 0.1% formic acid [A] and acetonitrile [B] HPLC system: Agilent 1290 HPLC system with 1290 DAD connected to QTOF Column: Polaris 3 C18, 150 × 2 mm, 3 μm (Varian) column thermostat: 30℃ Flow rate: 0.3 ml/min Injection volume: 2 μL Wavelength: 385 nm for measuring compound 1A /AMB, scanning 190-600 nm Software: MassHunter qualitative analysis B. 0.600 post time: 5 min to operate QTOF in negative electrospray mode (ESI-)

The HPLC gradient is shown in Table 1. Table 1 HPLC gradient: Time (min) Mobile phase B% 0 4.8 20 61.8 twenty two 80.0 22.5 4.8 23.0 Finish

Size and size distribution Nanoparticle size, size distribution, dry weight and drug loading are summarized in Table 2. Table 2 Examples of formulations 1 2 3 Z average diameter (nm) ¹ 143 158 214 Average number average diameter (nm) ¹ 85 48 40 Size polydispersity index (PDI) ¹ 0.18 0.25 0.33 Number average (mode) diameter (nm) ² 74 78 82 Number average (average) diameter (nm) ² 87 103 64 Dry weight (g) or (mg/mL) ³ 0.071 g 2.14 mg/mL 1.04 mg/mL Compound 1A loading amount [% (w/w)] 1.07 2.8 3.9 ¹ As measured by Zetasizer; ² As measured by NTA; ³ Example 1 particles were lyophilized, and Example 2 and 3 particles were dialyzed to produce dry material (g) and aqueous suspension (mg) /mL).

Table 3 provides details of the particle size distribution measured by NTA. Table 3 Examples of formulations 1 2 3 Average diameter (nm) 87.3 103 82.4 Mode diameter (nm) 73.5 78 64.6 Standard deviation (nm) 22.2 35.8 30.6 D10 (nm) 64 71 57.4 D50 (nm) 83.1 92.9 73.4 D90 (nm) 114.4 152.3 118

Drug loading and stability In the composition from Example 1, 42 mol% of the polyene in the particles is compound 1A . The loading of compound 1A is 1.1% (w/w), and the total polyene loading is 2.5% (w/w).

It is found that the composition from Example 2 contains the same polyene impurities as in Example 1, but with a lower content. This is because 10% of the total polyenes corresponds to the same main polyene impurities found in Example 1. . The concentration of compound 1A in the liquid sample is 0.59 mg/mL, so that the loading amount of compound 1A in the particles is 2.8% (w/w).

In the composition of Example 3, no significant degradation of compound 1A was observed. The concentration of compound 1A in the liquid sample is 0.40 mg/mL, so that the loading of compound 1A in the particles is 3.9% (w/w). Although a small amount of tiny polyene impurities can be observed, its concentration is too low to determine its quality. Poly (2-ethylhexyl cyanoacrylate) long-term stability of the nanoparticles in the compound 1A of the two weeks before analysis after preparation Example 3 compositions. With UV at λ = 385 nm, no degradation due to storage at 4°C for two weeks was observed.

A longer period of time and different temperature and humidity levels can be used for the storage period of the test composition.

Example 12. Efficacy of the composition of Example 3 The efficacy of the composition was determined using the lowest inhibitory concentration analysis, and the concentration of the 50% growth-inhibiting active compound (MIC50) given to the target organism was reported. The analysis was performed in a well plate with two (Müller-Hinton medium) or three parallel (M19-medium) cell cultures for each condition.

Cell culture medium : Ma-Xin Ershi and M19 without NaCl. Indicator organism : Stock solution of Candida albicans ATCC 10231 active compound and control : • Amphotericin B (USP): After inoculating 2.5 μg/mL in the well at the highest concentration, dissolve the vacuum-dried powder in DMSO until 2.5 mg /mL to get the final concentration. • Compound 1A (customized): After inoculating 2.5 μg/mL compound 1A in the wells at the highest concentration, dissolve the powder in DMSO up to 2.5 mg/mL to obtain the final concentration. Assume that the batch is 70% pure. • Example 3 composition: The formulation is a suspension with 18 mg/mL nanoparticles. The suspension was diluted in the culture medium and Candida albicans inoculum to a final concentration of 5 μg/mL compound 1A and 130 μg/mL poly(2-ethylhexyl cyanoacrylate). Prepare 10 dilutions of this solution to obtain the lowest concentration of 0.009 µg/ml compound 1A . • Empty poly(ethyl butyl cyanoacrylate) particles: Empty poly(ethyl butyl cyanoacrylate) particles are used as a reference. Hollow poly(ethyl butyl cyanoacrylate) was produced in the same manner as in Examples 2 and 3, except that it was prepared at pH 1 and the vanilla extract concentration was 10% (w/w). The particles were diluted in the same manner as in Example 3. The highest poly(ethylbutyl cyanoacrylate) concentration tested was 130 μg/mL.

Perform growth measurement under OD600.

As described in Example 11, Compound 1A was found to be stable in the Example 3 composition. This material was tested in an in vitro efficacy analysis against Candida albicans to verify that the active compound is released from the particles at a rate sufficient to inhibit Candida albicans to the same extent as pure compound 1A.

The MIC50 value measured in M19 medium is higher than the MIC50 value measured in Ma-Sin's medium because the strain grows faster in M19-medium. Manual measurement disk all night. Both analyses in M19 medium and Ma-Sin's medium showed that the MIC50 of the composition of Example 3 was 2-2.5 times higher than the MIC50 of pure compound 1A. As a control, the inhibition of empty poly(ethylbutyl cyanoacrylate) particles was tested in M19 medium. No growth inhibition of Candida albicans ATCC 10231 was observed up to a concentration of 130 μg/mL poly(ethylbutyl cyanoacrylate), which was the highest concentration tested. The highest test concentration of Example 3 composition (5 μg/mL) contained 130 μg/ml poly(2-ethylhexyl cyanoacrylate). Table 4. MIC50 (μg/mL) M19 medium Ma - Xin's medium Amphotericin B 0.34 0.04 Example 3 0.38 0.15 Compound 1A 0.18 0.06

Example 13 : Generation and characterization of dye-loaded nanoparticles: Preparation of other nanoparticles including compound 1 or a pharmaceutically acceptable salt thereof (for example, compound 1A or a pharmaceutically acceptable salt thereof). For example, the following microemulsion method is used to prepare PEG-coated and dye-loaded nanoparticles of polymers or lipids: Polymer Nanoparticles: Prepared from alkyl cyanoacrylate (such as n-butyl cyanoacrylate) ), an oil phase composed of a mixture of a pharmaceutically acceptable oil (such as miglitol) and the added near-infrared dye (such as DIR). Subsequently, an aqueous phase containing one or more surfactants (for example, Bridger L23 and/or Cleaver HS 15) is added to the oil phase. A specific surfactant such as Cliff HS 15 can be used as a polymerization initiator, thus producing a polymer excipient (e.g., poly(alkyl cyanoacrylate)) that is covalently linked to polyethylene glycol. The oil-in-water emulsion is prepared by mixing the oil and water phases. The dispersion is dialyzed with an aqueous fluid (for example, with a Spectra/Por dialysis membrane MWCO 100,000 Da) to remove the surfactant that is not incorporated in the nanoparticles. Lipid Nanoparticles: For a mixture of lipids (such as stearic acid and isopropyl palmitate) and a mixture of pharmaceutically acceptable oils (such as Miglitol) and added near-infrared dyes (such as DIR) The lipid phase of the composition is preheated until it melts. Prepare an aqueous phase consisting of distilled water and additives (such as surfactants (such as Lecithin 80H and Andean QDP Ultra)). Mix the lipid and water phase. Exemplary production and characterization of nanoparticles loaded with compound 1A : Preparation of poly(alkyl cyanoacrylate) nanoparticles coated with PEG and loaded with compound 1A by the following microemulsion method: preparation of containing alkyl cyanoacrylate (E.g. 2-ethylbutyl cyanoacrylate), a pharmaceutically acceptable oil (e.g. Miglitol) and the oil phase of compound 1A. Prepare an aqueous phase containing surfactants (such as Bridger L23 and Clifford HS 15). Cliff HS 15 can also act as a polymerization initiator. The oil-in-water emulsion is prepared by mixing the oil and water phases. The dispersion is deeply dialyzed with an aqueous fluid to remove surfactants that are not associated with the particles (for example, with a dialysis membrane, MWCO 100,000 Da).

Dynamic light scattering (such as Zetasizer) can be used to determine hydrodynamic diameter, hydrodynamic diameter distribution, and zeta potential. The dry weight content (nanoparticle concentration) of the final solution can be determined after sufficient drying. In order to calculate the amount of encapsulated drug, the drug content was extracted from the particles, and the extracted amount of compound 1A was quantified by using the LC-MS/MS method. The dynamic light scattering method usually displays the nanoparticle size (z-average) of the drug-loaded nanoparticle.

Generation and characterization of microbubbles stabilized by nanoparticle: The gas-filled microbubbles associated with the nanoparticle are generated as follows: a solution containing surface active compounds (for example, 2% (w/w) casein) is prepared. The compound 1A- loaded PEGylated nanoparticles (for example, Examples 2, 3, and 8-10 or the nanoparticles described in this example) were mixed with the casein solution. Saturate the solution with a gas (e.g. air or perfluoropropane). Seal the vial with a septum under an aerated atmosphere. In some preparation methods for microbubble-containing formulations, the composition can be degassed before adding microbubble gas.

The average size and concentration of the microbubbles stabilized by the nanoparticles were measured from the light microscope image using a 20× phase contrast objective lens and a cell counter (hemocytometer). Count the microbubbles and calculate the size by analyzing the image with ImageJ image analyzer.

Use fluorescence microscopy (using the same type of nanoparticles that only encapsulate fluorescent dyes instead of drugs) and electron microscopy to confirm that nanoparticles and microbubbles are associated to form a stable (single) layer.

Example 14 In this study, for example, in healthy animals (e.g., mice), the potential of microbubbles associated with nanoparticles for delivery of specific drugs to the lung was evaluated. High local concentration is achieved with gas-filled microbubbles stabilized by nanoparticles.

method The study was designed to have one animal in each group. Nanoparticles labeled with near-infrared fluorescent dyes were developed according to the procedure described in Example 13 and used. Use the complete animal imager to locate these nanoparticles inside the small animal.

In the experiment, if the test animal needs to be sacrificed, the animal can be anesthetized, used in the experiment, and sacrificed before waking up. During the storage period in the animal facility, the welfare of the animals is monitored, and the animals are given food and water at will.

Animal experiment: 1. Randomly select animals, weigh and give a solution that provides complete anesthesia (for example, fentanyl/medetomidine/midazolam/water (2:1:2:5) ) Of subcutaneous injection. 2. Place the catheter and allow intravenous injection. 3. Air bubbles required for injection. 4. Put the animal to sleep for the required time, and then euthanize it. 5. The lung, liver, kidney and spleen can then be collected. 6. A complete animal imager (such as Pearl) can be used to image fluorescence from organs.

Nanoparticles: In order to perform animal experiments, use near-infrared labeled nanoparticles.

After a control containing only nanoparticles, a pharmaceutical composition comprising nanoparticles associated with microbubbles (e.g. as described in Example 13) was tested, where the microbubbles contained a gas (e.g. perfluoropropane).

In order to directly compare the results, the lungs from a plurality of animals (e.g., three) are imaged together.

Microbubbles with lipid nanoparticles: Generate and test microbubbles with lipid nanoparticles.

The stability of lung accumulation is tested to assess whether nanoparticles remain in the lung or redistribute to other organs. Subsequently, the biodistribution in the animals (e.g., two) after the required amount of time (e.g., 1 h and 2 h after injection) is checked.

The high local concentration of nanoparticles in the lung is beneficial for targeted delivery of active pharmaceutical ingredients to lung tissue.

Example 15 In this study, gas-filled microbubbles stabilized by nanoparticles were tested in targeted drug delivery to the lung. For example, performing research in healthy mice.

method Nanoparticles labeled with fluorescent dyes (such as near-infrared dyes) were prepared as described in Example 13. Use the complete animal imager to locate these nanoparticles inside the small animal.

In the experiment, if the test animal needs to be sacrificed, the animal can be anesthetized, used in the experiment, and sacrificed before waking up.

The generation of microbubbles stabilized by nanoparticle: The gas-filled microbubbles associated with the nanoparticle are generated as follows:

Prepare a solution containing surface active compounds (eg 2% (w/w) casein). The dye-loaded PEGylated NP is mixed with the casein solution. Saturate the solution with a gas (such as sulfur hexafluoride or perfluoropropane). Modifiers (such as urea) are added at once to further promote the association between microbubbles and nanoparticles. Seal the vial with a septum under an aerated atmosphere. Animal experiments were performed as described in Example 10. Each animal is injected intravenously, for example, any of the following: A) The perfluoropropane-filled microbubbles are stabilized by poly(2-ethylhexylcyanoacrylate) nanoparticles. B) Perfluoropropane filled microbubbles with urea stabilized by poly(2-ethylhexyl cyanoacrylate) nanoparticles. C) The microbubbles filled with sulfur hexafluoride are stabilized by poly(2-ethylhexyl cyanoacrylate) nanoparticles.

After the injection, images were acquired, and the fluorescence intensity was used to evaluate the biodistribution of the drug-filled nanoparticles in the lungs of the test animals.

Example 16 As measured by the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC), the efficacy of compound 1A was tested against a wide range of yeast and filamentous fungal strains. Tested as comparators Amphotericin B, Caspofungin, Fluconazole and Voriconazole.

Materials: The isolated strains were obtained from the Culture Collection at the Center for Medical Mycology and included 20 strains of each of the following: Candida albicans, Candida glabrata (C. glabrata ), Subsidiary C. parapsilosis , C. tropicalis , Cryptococcus neoformans , Aspergillus fumigatus , A. niger , A. terreus ), Fusarium and mucormycete ( Rhizopus spp. and Mucor spp. ). It also includes 10 strains of each of the genera C. krusei, Aspergillus sp. and Penicillium, and 5 strains of Paecilomyces. This group contains 17 Candida strains with known MICs that are high compared to other current antifungal agents; it also includes the latest clinical strains. The test also included 15 dimorphic fungal isolates.

Minimum inhibitory concentration: Perform MIC test according to the clinical and Laboratory Standards Institute (CLSI) M27-A3 and M38-A2 standards for the susceptibility test of yeast and filamentous fungi (to isolate Cryptococcus The plant was cultivated for 72 h). For yeast strains, the incubation temperature and time are 35°C and 24-48 h, respectively, and the size of the inoculum is 0.5-2.5 × 10 ³ CFU/ml. For filamentous strains, the size of the inoculum is 0.4-5 × 10 ⁴ CFU/ml, and the incubation time is specific to the strain and drug. With the exception of YNB for Cryptococcus, RPMI 1640 is always the test medium. After 24 h and 48 h incubation, record the inhibitory endpoint of compound 1A at 50% and 100%; read the result of caspofungin against the aspergillus strain as the minimum effective concentration (MEC), that is, compared to the control The lowest concentration that causes small, round, compact growth in terms of confluent growth.

Minimum fungicidal concentration: The MFC determination was performed according to the modification previously described by: Canton et al., Diagn. Microbiol. Infect. Dis. , 45:203-206, 2003 and Ghannoum and Isham, Infectious Diseases in Clinical Practice , 15(4 ): 250-253, 2007. Specifically, the total content of each transparent well from the MIC analysis was subcultured onto potato dextrose agar. In order to avoid antifungal residues, the aliquot was soaked in agar and then once dried, it was streaked to separate, thus removing the cells from the drug source. Fungicidal activity is defined as a decrease of ≥ 99.9% in the number of colony forming units (CFU) per milliliter relative to the initial inoculum count, and fungicidal activity is defined as a decrease of <99.9%. If the MFC/MIC ratio of the drug is ≤ 4, it is considered fungicidal, or if the ratio is> 4, it is considered fungicidal. If the MIC and MFC values are within 2 dilutions, they are considered equivalent.

Endpoint determination: Compared with the growth control, all compound 1A MIC endpoints were recorded at both 50% and 100% inhibition. Although compound 1A did show both 50% and 100% inhibition against all Paecilomyces and Candida parapsilosis strains, all MIC values at the endpoint of 100% inhibition are reported below.

Incubation time determination: The growth inhibition after exposure to compound 1A was recorded after 24 hours and 48 hours of incubation. Among the exceptions were Mucor, which was read only at 24 h due to the rapid growth rate; and Cryptococcus strains, which were read after 72 h to be consistent with the incubation period of all other comparisons. In any of the tested species, no difference was found between the MIC values recorded at 24 h and 48 h. However, since some strains of Penicillium and Paecilomyces showed no visible growth at 24 hours in the MIC analysis, all MIC values at the 48-h incubation time point are reported below.

Results MIC and MFC determination of Candida strains: Table 5 shows the MIC and MFC data of Compound 1A and the comparatives against Candida albicans. These Candida albicans include fluconazole-susceptible strains (n = 13) and fluoride Conazol resistant strains (n=7) both. MIC _{50 is} defined as the lowest concentration that inhibits 50% of the test strain and MIC _{90 is} defined as the lowest concentration that inhibits 90% of the test strain. MFC _{50 is} defined as the lowest concentration that kills 50% of the tested strains and MFC _{90 is} defined as the lowest concentration that kills 90% of the tested strains. Table 5. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Fluconazole resistance n = 7 Scope 1-2 1->4 0.25-1 0.5-1 0.125-2 0.5-＞32 32-＞32 ＞32 ＜0.06-＞32 16-＞32 MIC/MFC ₅₀ N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MIC/MFC ₉₀ N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Fluconazole susceptibility n = 13 Scope 0.125-1 0.125-1 0.125-0.5 0.125-1 0.125-0.25 0.5-32 0.125-0.5 8-＞32 ＜0.06-0.06 ＞32 MIC/MFC ₅₀ 0.5 0.5 0.25 0.25 0.25 0.25 0.25 ＞32 0.6 ＞32 MIC/MFC ₉₀ 1 1 0.5 1 0.25 2 0.25 ＞32 0.06 ＞32

Table 6 shows the fungicidal activity of compound 1A and the comparatives against Candida glabrata strains. Table 6. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.25-2 0.5-2 0.125-1 0.5-1 0.25-8 1-＞32 2->64 ＞64 0.06->32 ＞32 MIC/MFC ₅₀ 1 1 0.5 0.5 0.5 1 8 ＞64 0.25 ＞32 MIC/MFC ₉₀ 2 2 0.5 1 2 32 ＞64 ＞64 4 ＞32

Table 7 shows the MIC and MFC data of all drugs against Candida krusei strains. Table 7. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-2 0.5-2 0.5-1 1 0.5-16 2->32 2-32 ＞64 ＜0.06-0.5 2->8 MIC/MFC ₅₀ 1 1 0.5 1 1 2 16 ＞64 0.125 ＞8 MIC/MFC ₉₀ 2 2 1 1 4 8 32 ＞64 0.25 ＞8

Table 8 shows the fungicidal activity against Candida parapsilosis. Table 8. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.25-2 0.25-2 0.125-1 0.5-2 0.25-2 2->32 0.25->64 ＞64 ＜0.06-＞32 1-＞32 MIC/MFC ₅₀ 0.25 0.5 0.25 1 0.5 ＞32 0.5 ＞64 ＜0.06 ＞32 MIC/MFC ₉₀ 2 2 0.5 1 2 ＞32 0.5 ＞64 0.06 ＞32

Table 9 shows the fungicidal activity of Compound 1A and the comparative substances against Candida tropicalis. Table 9. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-8 0.5-16 0.25-0.5 0.25-1 0.06-2 0.5-＞32 0.25-2 ＞64 ＜0.06-1 ＞32 MIC/MFC ₅₀ 1 1 0.25 0.5 0.25 32 0.25 ＞64 0.06 ＞32 MIC/MFC ₉₀ 8 8 0.5 1 0.5 ＞32 0.5 ＞64 0.06 ＞32

Table 10 summarizes the MIC and MFC data of compound 1A and the comparator against all Candida strains. Table 10. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.125-8 0.125-16 0.125-1 0.125-2 0.06-16 0.5-＞32 0.125->32 8-＞32 ＜0.06-＞32 1-＞32 MIC/MFC ₅₀ 1 1 0.5 0.5 0.5 16 0.5 ＞32 0.06 ＞32 MIC/MFC ₉₀ 2 2 0.5 1 2 ＞32 ＞32 ＞32 1 ＞32

MIC and MFC determinations against Cryptococcus strains Table 11 shows the activity of compound 1A and its comparator against Cryptococcus neoformans strains. Table 11. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-2 0.5-＞4 0.06-1 0.25-2 1-16 16-＞32 ＜0.06-16 2 ＜0.06-0.25 0.06->0.5 MIC/MFC ₅₀ 1 1 0.25 0.5 16 ＞32 2 ＞64 ＜0.06 ＞0.5 MIC/MFC ₉₀ 1 ＞4 0.5 1 16 ＞32 8 ＞64 0.125 ＞0.5

Determination of MIC and MFC for Aspergillus strains Tables 12-15 show the MIC and MFC data for compound 1A and comparative substances of individual Aspergillus genus. Table 16 is a summary of the MIC and MFC data of all tested Koji bacteria strains. Table 12. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 1-4 2->4 1-8 1-＞32 0.125-8 8-＞32 32->64 ＞64 0.25-4 1-＞16 MIC/MFC ₅₀ 2 4 8 8 0.25 ＞32 ＞64 ＞64 1 4 MIC/MFC ₉₀ 2 ＞4 8 ＞32 0.5 ＞32 ＞64 ＞64 2 ＞16 Table 13. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 1-2 1->4 1-2 1-＞8 0.25-1 32-＞32 64 64 0.25-2 1-＞8 MIC/MFC ₅₀ 1 2 1 ＞8 0.25 ＞32 64 64 0.5 8 MIC/MFC ₉₀ 2 ＞4 2 ＞8 0.5 ＞32 64 64 1 ＞8 Table 14. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 1-2 2->4 0.25-2 0.25-＞8 0.125-2 16-＞32 32->64 ＞64 0.06-8 0.5-＞8 MIC/MFC ₅₀ 2 2 1 2 0.125 ＞32 ＞64 ＞64 1 ＞8 MIC/MFC ₉₀ 2 4 1 ＞8 0.25 ＞32 ＞64 ＞64 4 ＞8 Table 15. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-4 2->4 0.25-16 2->32 0.25-1 32-＞32 4->64 ＞64 0.25-2 1->4 MIC/MFC ₅₀ 2 ＞4 8 ＞32 0.25 ＞32 64 ＞64 0.5 ＞4 MIC/MFC ₉₀ 4 ＞4 8 ＞32 0.5 ＞32 ＞64 ＞64 0.5 ＞4 Table 16. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-4 1->4 0.25-16 0.25-＞8 0.125-8 8-＞32 4->64 ＞64 0.06-8 0.5-＞8 MIC/MFC ₅₀ 2 4 1 ＞8 0.25 ＞32 ＞64 ＞64 0.5 ＞8 MIC/MFC ₉₀ 2 ＞4 8 ＞8 0.5 ＞32 ＞64 ＞64 2 ＞8

Measurement of MIC and MFC for difficult/rare fungi Table 17 shows the MIC and MFC data of Fusarium strains. Table 17. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 1-4 1-4 0.5-2 1-4 1-64 ＞32 8->64 ＞64 2->32 8-＞32 MIC/MFC ₅₀ 2 2 1 2 32 ＞32 ＞64 ＞64 16 32 MIC/MFC ₉₀ 4 ＞4 2 2 32 ＞32 ＞64 ＞64 ＞32 ＞32

Table 18 shows the MIC and MFC data of Fusarium strains. Table 18. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 1-4 1->4 0.25-2 0.5-＞8 32-＞32 ＞32 ＞32 ＞32 8-＞16 16-＞16 MIC/MFC ₅₀ 2 2 0.5 1 ＞32 ＞32 ＞32 ＞32 16 ＞16 MIC/MFC ₉₀ 4 ＞4 2 8 ＞32 ＞32 ＞32 ＞32 ＞16 ＞16

The results for Penicillium and Paecilomyces strains can be seen in Tables 19 and 20, respectively. Table 20 only contains the concentration range. This is because the number of Paecilomyces strains (5) is too small to calculate the MIC ₅₀ and MIC ₉₀ values. Table 19. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-2 2->4 0.5-4 1-＞8 0.25->32 16-＞32 1->64 64->64 0.5-＞32 1-＞32 MIC/MFC ₅₀ 2 2 1 4 4 ＞32 ＞64 ＞64 4 ＞32 MIC/MFC ₉₀ 2 4 2 ＞8 32 ＞32 ＞64 ＞64 ＞32 ＞32 Table 20. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope 0.5-＞4 4->4 0.25-＞8 ＞4 1-＞32 32-＞32 32->64 ＞64 0.125-2 1-＞8 MIC/MFC ₅₀ N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MIC/MFC ₉₀ N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

MIC and MFC Measurements for Type II Fungi Table 21 shows the MIC data of Blastomyces dermatitidis , Coccidioides immitis and Histoplasma capsulatum strains. MFC is not performed on these fungi, because these fungi are limited fungi, and there is no standardized method for performing MFC for them. Table 21. 1A MIC 1A MFC Ambibacterium MIC Ambibacterium MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC Scope ＜0.03-0.25 ND ＜ 0.03-0.125 ND 0.03-0.5 ND 2-32 ND ＜ 0.03-0.125 ND ND = Not determined due to lack of standardized methods

Comparison of MIC and MFC determinations against other Candida strains with higher MIC relative to other antifungal agents MIC data of compound 1A. Table 22. species 1A MIC 1A MFC Caspofungin MIC Caspofungin MFC Fluconazole MIC Fluconazole MFC Voriconazole MIC Voriconazole MFC White 2 2 ＞64 ＞64 32 ＞32 White 1 1 ＞32 ＞32 White 1 1 ＞32 ＞32 White 1 1 32 ＞32 White 1 1 1 32 ＞32 ＞32 White 1 1 1 ＞32 32 ＞32 ＞0.06 16 White 1 ＞4 2 ＞32 ＞32 ＞32 0.5 ＞32 smooth 1 1 2 32 32 ＞64 1 ＞32 smooth 1 1 1 32 2 ＞64 ＞32 ＞32 smooth 1 1 ＞64 ＞64 1 ＞32 smooth 1 1 2 16 ＞64 ＞64 1 ＞32 smooth 1 1 4 ＞32 8 ＞64 0.25 ＞32 smooth 2 2 8 32 64 ＞64 2 ＞32 Cruise 0.5 2 16 ＞32 16 ＞64 Near smooth 0.5 0.5 2 ＞32 0.25 ＞64 0.06 ＞32 Near smooth 0.5 0.5 2 ＞32 0.5 ＞64 0.06 ＞32 Near smooth 1 1 2 ＞32 1 ＞64 ＞32 ＞32

Example 17 In the treatment of disseminated aspergillosis in a murine model of immune insufficiency, the efficacy of Compound 1A was tested and compared with Ambimycin, Voriconazole and Caspofungin.

Female CD-1 mice (Charles River Laboratories, Wilmington, MA) of approximately 30 g each were used as a model. Set the environmental control of the animal room to maintain a temperature of 16°C to 22°C, a relative humidity of 30%-70%, and a 12:12 light-dark cycle.

Preparation of standard inoculum organisms: Aspergillus fumigatus AF9l was obtained from the Culture Collection of the CWRU Medical Mycology Center. Subculture the cells from frozen stock on potato dextrose agar (PDA) plates. Subsequently, the cells were collected using sterile saline with 0.05% Tween 80, centrifuged, and washed three times with physiological saline (0.85% NaCl). Use a hemocytometer to prepare 1 × 10 ⁷ challenge inoculums.

Verification of the inoculum count: In order to check the inoculum count, spread the ten-fold dilution of the conidia suspension treated with Aspergillus fumigatus on the PDA medium. The plates were incubated at 37°C for 2-4 days and the colony counts were determined.

Immunosuppression: Mice received subcutaneous cyclophosphamide at the following doses: 150 mg/kg 4 days before infection, 100 mg/kg 1 day before infection, and 100 mg/kg 2 days after vaccination. On the day of the challenge, blood was collected from one of the mice from each group for white blood cell count to verify immunosuppression.

Infection: Each mouse was challenged (via the tail vein) with 0.1 ml normal saline containing 1 × 10 ^{7 conidia.} The animal is considered infected after successful IV administration of the inoculum and confirmation of the inoculum (see section 7d). Using tissue fungal burden and survival as indicators, 5 mice/group (randomly selected) for tissue fungal burden and 10 mice/group for survival were used to evaluate the efficacy of the treatment group and the control group.

Test compound: The sponsor provided a test article, namely compound 1A (batch ELN EXP-11-AJ1675, potency 928 mg/g). It is administered intravenously in a 5% dimethylsulfoxide (DMSO) solution containing 5% aqueous glucose. The solution is prepared from a stock solution of compound 1A in DMSO stored at -20°C on the day of use. The comparators (Ampicillin, Voriconazole and Caspofungin) were purchased from the pharmacy by the Medical Mycology Center. Dissolve it in sterile water according to the manufacturer's instructions, and obtain an aliquot containing the selected dose.

In this report, the dose and concentration of Ambiont are expressed as the content of Amphotericin B.

Treatment group: The infected mice were randomly divided into the following groups (for tissue fungal burden: 5 animals/group and for survival: 10 animals/group).

Experiment I-The treatment groups are as follows: 1.0 mg/kg compound 1A , 0.5 mg/kg compound 1A , 7.5 mg/kg ambiont, 3.5 mg/kg amblyt, 7.5 mg/kg voriconazole, 1.0 mg/kg caspofene Net, vehicle (5% DMSO containing 5% aqueous glucose); and untreated control group.

Experiment II-The treatment group is as follows: 1.0 mg/kg Compound 1A , 0.5 mg/kg Compound 1A , 1.0 mg/kg Ambidium, 0.5 mg/kg Ambidium; and untreated control group. All treatments are given intravenously.

Treatment schedule: starting two hours after inoculation, the animals were treated for a period of seven days. Compound 1A and caspofungin were administered once a day, and Ambhitin was administered every other day in Experiment I and every day in Experiment II. Because of the rapid clearance of voriconazole, it was given twice a day with an interval of 8 hours.

Tissue fungal burden: The mice were sacrificed one day after the last day of treatment; the kidneys and lungs were then aseptically removed and weighed. The tissue is homogenized and serially diluted in phosphate buffered saline. The homogenate was cultured on the PDA plate for 48 h to determine the colony forming unit (CFU); the tissue fungal burden was expressed as CFU/gram of tissue.

Survival analysis: Monitor infected mice and record any signs of disease (ie lethargy, weight loss, general growth retardation) or death twice a day until 28 days after vaccination. The average weight of each treatment group was also recorded every day. Euthanize dying animals that cannot eat/drink.

Statistical analysis: Kaplan-Meier was used to compare survival differences, and a non-parametric independent Mann-Whitney statistical test was used to compare the average CFU in the kidney or lung. A P value of <0.05 is regarded as statistically significant.

Results in vitro activity: Table 23 shows the in vitro activity of compound 1A and comparative agents and amphotericin B against K. fumigatus AF9 l (infected strain). Table 23 1A Amphimurium Amphotericin B Voriconazole Caspofungin MIC (μg/mL) 2 4 2 1 0.25 (MEC) MFC (μg/mL) 2 N/A 16 ＞ 8 ＞ 16

Experiment I Survival: In Figure 1, survival is given as a percentage of the total number of animals in a group on the first day of treatment.

Kidney tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 24). The renal fungal burden is analyzed and is given as the mean log CFU ± standard deviation.

Lung tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 24). The lung fungal burden is analyzed and given as the mean log CFU ± standard deviation. Table 24. Average log CFU ± standard deviation treat kidney lung 0.5 mg/kg 1A 3.16 ± 0.40 3.46 ± 0.49 1.0 mg/kg 1A 0.51 ± 1.02 0.55 ± 1.10 3.5 mg/kg Ambibacterium 0.45 ± 1.00 0.47 ± 1.06 7.5 mg/kg Ampi 0.98 ± 1.34 1.11 ± 1.52 7.5 mg/kg voriconazole 3.19 ± 0.59 3.38 ± 0.45 1.0 mg/kg caspofungin 3.11 ± 0.62 3.41 ± 0.64 Vehicle control 3.40 ± 0.18 3.56 ± 0.19 Untreated control 3.34 ± 0.39 3.52 ± 0.20

Experiment II Survival: As can be seen in Figure 2, survival is given as a percentage relative to the total number of animals in a group on the first day of treatment.

Kidney tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 25). The renal fungal burden is analyzed and is given as the mean log CFU ± standard deviation.

Lung tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 25). The lung fungal burden is analyzed and given as the mean log CFU ± standard deviation. Table 25. Average log CFU ± standard deviation treat kidney lung 0.5 mg/kg 1A 2.93 ± 0.73 2.75 ± 0.93 1.0 mg/kg 1A 1.16 ± 1.10* 1.17 ± 1.15* 0.5 mg/kg Ampi 3.13 ± 0.94 2.37 ± 0.17 1.0 mg/kg Ambibacterium 2.72 ± 1.06 2.26 ± 0.34 Untreated control 3.31 ± 0.37 3.30 ± 0.25 *When compared with the untreated control group, the 0.5 mg/kg Ambibacterium treatment group, and the 0.5 mg/kg 1A treatment group, the P value is less than 0.05.

Example 18 In the treatment of disseminated candidiasis in a murine model of immune insufficiency, the efficacy of compound 1A was evaluated compared with ambimol, voriconazole, fluconazole and caspofungin.

Female BALB/c mice (Charles River Laboratories, Wilmington, MA) of approximately 20 g each were used as a model. Set the environmental control of the animal room to maintain a temperature of 16°C to 22°C, a relative humidity of 30%-70%, and a 12:12 light-dark cycle.

Preparation of standard inoculum organisms: The clinical Candida albicans SC5314 strain was obtained from the CMM Culture Collection and used as an infectious fungus. Candida albicans was spread on Sabouraud dextrose agar (SDA) and incubated at 37°C for 2 days. The Candida albicans cells were collected by centrifugation and washed with saline (0.85% NaCl). Prepare 5 × 10 ⁵ challenge inoculums using a hemocytometer.

Verification of the inoculum count: In order to check the inoculum count, a ten-fold dilution of the Candida albicans-treated conidia suspension was spread on the SDA medium. The plate was incubated at 37°C for 2 days and the colony count was determined.

Immunosuppression: Mice received subcutaneous cyclophosphamide at the following doses: 150 mg/kg 4 days before infection, 100 mg/kg 1 day before infection, and 100 mg/kg 2 days after vaccination. On the day of the challenge, blood was collected from one of the mice from each group for white blood cell count to verify immunosuppression.

Infection: each mouse was challenged (via the tail vein) with 0.1 ml normal saline containing 1 × 10 ^{4 spores.} The animal is considered infected after successful IV administration of the inoculum and confirmation of the inoculum. Using tissue fungal load and survival as indicators, 5 mice/group for tissue fungal load and 10 mice/group for survival were used to evaluate the efficacy of the treatment group and the control group. The tissue load group and survival group of Experiment I were performed at two different times.

Test compound: The sponsor provided a test article, namely compound 1A (batch ELN Exp-11-AJ1675, potency 928 mg/g). It is administered intravenously in a 5% dimethylsulfoxide (DMSO) solution containing 5% aqueous glucose. The solution is prepared from a stock solution of compound 1A in DMSO stored at -20°C on the day of use. Comparators (Apimycin, Voriconazole, Caspofungin and Fluconazole) were purchased from the pharmacy by the Center for Medical Mycology. Dissolve it in sterile water according to the manufacturer's instructions, and obtain an aliquot containing the selected dose.

In this report, the dose and concentration of Ambiont are expressed as the content of Amphotericin B.

Treatment group: The infected mice were randomly divided into the following groups (for tissue fungal burden: 5 animals/group and for survival: 10 animals/group).

Experiment I-The treatment groups are as follows: 0.7 mg/kg compound 1A , 0.35 mg/kg compound 1A , 5.4 mg/kg ambiont, 2.7 mg/kg amblyt, 4 mg/kg voriconazole, 0.35 mg/kg caspofene Net, 6 mg/kg fluconazole, vehicle (5% DMSO containing 5% aqueous glucose); and untreated control group.

Experiment II-The treatment group is as follows: 0.7 mg/kg Compound 1A , 0.35 mg/kg Compound 1A , 0.7 mg/kg Ambibacterium, 0.35 mg/kg Ammonium; and untreated control group. All treatments are given intravenously.

Treatment schedule: starting two hours after inoculation, the animals were treated for a period of seven days. With the exception of voriconazole, treatment is given once a day. Because of the rapid elimination of voriconazole, it is given twice a day with an interval of 8 hours.

Tissue fungal burden: The mice were sacrificed one day after the last day of treatment, the kidneys and brain were aseptically removed and weighed. The tissue is homogenized and serially diluted in phosphate buffered saline. The homogenate was cultured on the SDA plate for 48 hours to determine the colony forming unit (CFU); the tissue fungal burden was expressed as CFU/gram of tissue.

Survival analysis: Monitor infected mice and record any signs of disease (ie lethargy, weight loss, general growth retardation) or death twice a day until 28 days after vaccination. The average weight of each treatment group was also recorded every day. Euthanize dying animals that cannot eat/drink.

Statistical analysis: The non-parametric independent Mann-Whitney test was used to compare the difference in average log CFU in the kidney or brain. The Kaplan-Meier test was used to compare survival differences. A P value of <0.05 is regarded as statistically significant.

Results in vitro activity: Table 26 shows the in vitro activity of compound 1A , comparative agents and amphotericin B against Candida albicans SC5314 (infected strain). Table 26. 1A Amphimurium Amphotericin B Voriconazole Fluconazole Caspofungin MIC (μg/mL) 0.5 0.5 0.125 0.008 0.25 0.25 (MEC) MFC (μg/mL) 0.5 N/A 0.25 ＞ 32 ＞ 64 32

Experiment I Survival: In Figure 3, survival is given as a percentage of the total number of animals in a group on the first day of treatment.

Kidney tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 27). The renal fungal burden is analyzed and is given as the mean log CFU ± standard deviation.

Brain fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 27). The brain fungal burden is analyzed and given as the mean log CFU ± standard deviation. Table 27. Average log CFU ± standard deviation treat kidney brain 0.35 mg/kg 1A 3.16 ± 1.41 2.92 ± 1.21 0.7 mg/kg 1A 2.66 ± 2.11 3.59 ± 1.20 2.7 mg/kg Ambibacterium 0.59 ± 0.93 0.14 ± 0.31 5.4 mg/kg Ambibacterium 0.51 ± 2.76 2.01 ± 0.82 4 mg/kg voriconazole 3.45 ± 1.15 3.42 ± 2.30 0.35 mg/kg caspofungin 3.22 ± 1.26 1.22 ± 1.87 6 mg/kg fluconazole 3.87 ± 1.53 1.28 ± 1.95 Vehicle control 4.46 ± 0.56 3.39 ± 2.31 Untreated control 4.62 ± 0.59 3.48 ± 1.36

Experiment II Survival: In Figure 4, survival is given as a percentage relative to the total number of animals in a group on the first day of treatment.

Kidney tissue fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 28). The renal fungal burden is analyzed and is given as the mean log CFU ± standard deviation.

Brain fungal burden: Tissue fungal burden is assessed one day after the last treatment or immediately after death in the case of dying animals (Table 28). The brain fungal burden is analyzed and given as the mean log CFU ± standard deviation. Table 28. Average log CFU ± standard deviation treat kidney brain 0.35 mg/kg 1A 3.53 ± 0.63 3.55 ± 1.26 0.7 mg/kg 1A 2.99 ± 1.05 ^a,b 2.97 ± 2.04 0.35 mg/kg Ampi 4.33 ± 0.50 4.04 ± 0.31 0.7 mg/kg Ambibacterium 4.27 ± 0.67 3.84 ± 0.68 Untreated control 4.57 ± 0.22 4.19 ± 0.84 ^a When compared with the untreated control group, the P value is less than 0.05. ^b When compared with the treatment group with Ampicilin 0.35, the P value is less than 0.05.

Example 19 has produced a total of 74 batches of particles; 26 of these batches have been produced using PACA and 48 batches have been produced using poly(lactic-co-glycolic acid) (PLGA).

Diameter, polydispersity index (PDI) and stability in suspension have been used as criteria to evaluate particle quality. In particular, the prepared particles are characterized as well-performing, acceptable, and under-performing as follows: A well-performing particle formulation is a stable suspension with a z-average diameter of <200 nm, a PDI of <0.3, and no visible viscosities; Acceptable particle formulations are stable suspensions with PDI> 0.3l; and The poorly performing particle formulations are unstable suspensions that are prone to phase separation and sedimentation.

A summary of the formulations produced in this example is provided in Table 29. Table 29. Numbering Formulation Compound characteristics Numbering Formulation Compound characteristics 1 POPZ good performance 45 PEHCA Acceptable 2 PACA Poor performance 46 PLGA, 2% Plu F68, NMP Poor performance 3 PACA Acceptable 47 PLGA, 2% Plu F127, NMP good performance 4 PACA Poor performance 48 PLGA, 4% Plu F127, NMP good performance 5 PACA Acceptable 49 PLGA, 2% Tween 80, NMP good performance 6 PACA Acceptable 50 PLGA, 2% Plu F68, 50% NMP in DMSO Performed well in the new system, and performed poorly after dialysis 7 PACA Acceptable 51 PLGA, 2% Plu F127, 50% NMP in DMSO good performance 8 PACA Acceptable 52 PLGA, 4% Plu F127, 50% NMP in DMSO good performance 9 PACA Poor performance 53 PLGA, 2% Tween 80, 50% NMP in DMSO Performed well in the new system, and performed poorly after dialysis 10 PEHCA Poor performance 54 PLGA, 4% Plu F127, 50% NMP in DMSO Performed well in the new system, and performed poorly after dialysis 11 PEHCA Poor performance 55 Polymerization of PEHCA with 1% V65 and 20% Clifford Acceptable after centrifugation 12 PEHCA Poor performance 56 Polymerization of PEHCA with 5% V65 and 20% Clifford Acceptable after centrifugation 13 PEHCA Poor performance 57 Polymerization of PEHCA with 5% V65 and 20% Cleaver and 5% vanilla extract Poor performance 14 PEHCA pH 1 good performance 58 Polymerization of PEHCA with 5% V65 and 20% Cleaver, 5% vanilla extract and 10% compound 1A good performance 15 PEHCA pH 2 good performance 59 2% PVA, NMP, 5% compound 1A , 5% vanilla extract good performance 16 PEHCA pH 3 Poor performance 60 4% PVA, NMP, 5% compound 1A , 5% vanilla extract Poor performance 17 PEHCA pH 4 Poor performance 61 2% F68, NMP, 5% compound 1A , 5% vanilla extract Poor performance 18 PEHCA pH 4 Poor performance 62 2% PVA, NMP, 10% compound 1A Poor performance 19 PEHCA, 5% vanilla extract pH 2 Acceptable 63 4% PVA, NMP, 10% compound 1A Poor performance 20 PEHCA, 5% vanilla extract pH 2, acetone Acceptable 64 2% F68, NMP, 10% compound 1A Poor performance twenty one PEHCA, 5% vanilla extract pH 2, acetone Poor performance 65 PLGA, 2% Plu F127, vanilla extract, NMP good performance twenty two PEHCA, 5% vanilla extract pH 2 Acceptable 66 PLGA, 4% Plu F127, vanilla extract, NMP good performance twenty three PEHCA, 5% vanilla extract pH 2, acetone Poor performance 67 2% Tween 80, vanilla extract, NMP good performance twenty four PEHCA, 5% vanilla extract pH 2, acetone Poor performance 68 4% Tween 80, vanilla extract, NMP good performance 25 Compatibility Study Lipids 69 PLGA, 2% Plu F127, NMP good performance 26 Testing the nano assembly PLGA Poor performance 70 PLGA, 4% Plu F127, NMP good performance 27 Extended compatibility studies lipids and oils 71 2% Tween 80, NMP good performance 28 PLGA particle synthesis good performance 72 4% Tween 80, NMP good performance 29 PLGA, 2% PVA, NMP Acceptable 73 PLGA, 2% Plu F127, DMF Acceptable 30 PLGA, 1% Plu F68, NMP Poor performance 74 PLGA, 4% Plu F127, DMF Acceptable 31 PLGA, 2% PVA, 10% NMP in acetone Poor performance 75 2% Tween 80, DMF Acceptable 32 PLGA, 2% Plu F68, 10% NMP in acetone Poor performance 76 4% Tween 80, DMF Acceptable 33 PLGA, 1% Plu F68, 10% NMP in acetone Poor performance 77 PEHCA, vanilla extract, compound 1A , Cleaver good performance 34 PLGA Poor performance 78 PEHCA, 1,6-HBCA, vanilla extract, compound 1A , Cleaver good performance 35 PLGA Poor performance 79 PEHCA, vanilla extract, compound 1A , Cleaver, Bridger good performance 36 PLGA Poor performance 80 PEHCA, 1,6-HBCA, vanilla extract, compound 1A , Cleaver Poor performance 37 PLGA Poor performance 81 PEHCA, vanilla extract, compound 1A , Cleaver, Bridger Poor performance 38 PLGA Poor performance 82 PEHCA, vanilla extract, compound 1A , Cleaver, Bridger Poor performance 39 PLGA Poor performance 83 PEHCA, Revanquinone, Compound 1A , Cleaver, Bridger Poor performance 40 PLGA Poor performance 84 PEHCA, Revanquinone, Compound 1A , Cleaver, Bridger Poor performance 41 PLGA Poor performance 85 Methanol/chloroform, 2% Plu Poor performance 42 PEHCA Acceptable 86 Methanol/chloroform, 4% Plu Poor performance 43 PEHCA Poor performance 87 Methanol/chloroform, 2% Tween 80 Poor performance 44 PEHCA Acceptable 88 Methanol/chloroform, 4% Tween 80 Poor performance

Formulation 45 was prepared using the following components. water box quantity unit Clifford HS 15 0.048 g Bridger L23 0.048 g 10 mM HCl. pH 2 4.8 g 6-O-palmitoyl-L-ascorbic acid 0.0048 The organic phase 2-ethylhexyl cyanoacrylate 0.32 g Compound 1A 0.0384 g Vanilla extract 0.04 g Miglitol 812 0.0056 g total: 5.30 g

Formulation 49 was prepared using the following components. quantity unit The organic phase Compound 1A 0.005 g Poly(lactide-co-glycolide) 0.05 g N -Methyl-2-pyrrolidone 2.5 ml Water phase (pH 7) Tween 80 0,05 g DI water 2.5 ml

Formulation 73 was prepared using the following components. quantity unit The organic phase Compound 1A 0.0025 g Poly(lactide-co-glycolide) 0.05 g N , N -Dimethylformamide 2.5 ml Water phase (pH 7) Pluronic F127 0.05 g DIW 2.5 ml

The poly(lactide-co-glycolide) used in this example has a lactide-glycolide ratio of 50:50, is ester-capped, and has a weight average molecular weight of 38000-54000 Da.

PACA particles As described below, multiple parameter changes used to generate PACA particles.

Monomers: Different types of PACA monomers are used to encapsulate compound 1A . The goal is to dissolve compound 1A in the oil phase without starting the polymerization reaction. Poly(ethylhexyl cyanoacrylate) (PEHCA) worked with compound 1A . Poly(butyl cyanoacrylate) (PEBCA) was also tested. During these tests, it was found that compound 1A initiated the polymerization of PEBCA and the resulting particles did not meet the requirements.

pH: Particle polymerization at low pH (for example, pH = 1) produces particles with excellent characteristics (size, size distribution, and colloidal stability). Since compound 1A oxidizes at a low pH, a higher pH 4 is used. It was found that the retention of compound 1A in the oil phase during production reduces its sensitivity to pH. Test the lower pH level to determine whether the particles have a higher loading capacity and loading efficiency. LC-DAD-QTOF analysis of these formulations showed that no degradation occurred, and therefore pH is not a key parameter for the stability of compound 1A.

Surfactant: a mixture of Bridger L35 and Clifford HS15 is usually used throughout the tests described in this article. It was discovered that Cliffe initiated the polymerization process.

Stabilizer: Test the effect of ascorbic acid on the stability of compound 1A in solution. During the polymerization, ascorbic acid is added to the water phase. It was found that compound 1A was chemically dehydrated under high concentration of ascorbic acid. It was found that high concentrations of ascorbic acid reduced the stability of compound 1A.

Test the effect of vanilla extract on the stability of compound 1A. Perform a series of experiments with and without vanilla extract. In these experiments, it was found that vanilla extract helped compound 1A to dissolve. It has also been observed that the addition of vanilla extract reduces the stability of the compound 1A/acrylate suspension. The oil phase can increase the solubility of the compound 1A for a possibility of impact of this description, this system is easier to react with an acrylate monomer compound 1A as observed by dissolving the compound of 1A was dissolved compared with untreated.

Generally speaking, the composition of PACA particles contains: • Water phase: Bridger L35, Cliff HS15 and HCl for pH adjustment • Oil phase: Acrylic monomers (ethylhexyl cyanoacrylate, ethyl cyanoacrylate Butyl cyanoacrylate, 1-heptyl cyanoacrylate and 2-phenylethyl cyanoacrylate/butyl cyanoacrylate), Miglitol, vanilla extract, compound 1A . 1-Heptyl cyanoacrylate and 2-phenylethyl cyanoacrylate/butyl cyanoacrylate cannot dissolve compound 1A .

Key findings: • PACA particles can stabilize compound 1A (tested for 2 weeks) in suspension. • Vanilla extract has a dissolving effect on compound 1A. • PACA particles can be produced at low pH without causing unacceptable levels of compound 1A to decompose. • Ascorbic acid and 6-O-palmitoyl-L-ascorbic acid, water-soluble antioxidants failed to increase the stability of compound 1A at the tested ascorbic acid concentration.

The LC-UV trace of one of the best performing PACA formulations for compound 1A is shown in FIG. 5.

PLGA particles PLGA nanoparticles have been studied as a vehicle for compound 1A. PLGA has certain advantages over PACA. For example, the PLGA polymer is preformed. This means that polymer excipients do not involve reactivity-related issues. In addition, PLGA allows the use of organic solvents, thereby allowing compound 1A to be completely dissolved before encapsulation.

The method used to prepare the PLGA particles described herein is nanoprecipitation. This procedure involves slowly adding an organic phase (containing PLGA, compound 1A and other optional hydrophobic ingredients such as vanilla extract) to an aqueous solution containing a surfactant. Alternatively, microfluidics can be used, for example, by mixing a fixed ratio of organic and aqueous phases in a continuous stream flowing through a microfluidic channel.

During the initial testing of PLGA particles, the organic phase has been added to the aqueous solution under magnetically driven stirring. We tested four different surfactants at 1-3 different concentrations. In addition, we have tested various organic solvents and organic solvent mixtures. The test conditions are summarized in Table 30.

Table 30 provides an overview of the test conditions during PLGA generation. The percentage of compound 1A represents the theoretical maximum dry weight loading. Table 30. NMP NMP NMP, 5% vanilla extract 10% NMP in acetone 10% NMP in ethanol 10% NMP in DMSO 50% NMP in DMSO DMF 2% PVA 10% compound 1A ² - 5% compound 1A ¹ 10% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ - 4% PVA 10% compound 1A ³ - 5% compound 1A ³ - - - - - 1% F68 10% compound 1A ³ - - 10% compound 1A ³ - - - - 2% F68 10% compound 1A ² - 5% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ - 2% F127 10% compound 1A ¹ 5% compound 1A ¹ 5% compound 1A ¹ - 10% compound 1A ³ 10% compound 1A ³ 10% compound 1A ³ 5% compound 1A ⁴ 4% F127 10% compound 1A ¹ 5% compound 1A ¹ 5% compound 1A ¹ - - - - 5% compound 1A ⁴ 2% Tween 80 10% compound 1A ¹ 5% compound 1A ¹ 5% compound 1A ¹ - - - - 5% compound 1A ⁴ 4% Tween 80 5% compound 1A ⁴ 5% compound 1A ¹ 5% compound 1A ¹ - - - - 5% compound 1A ^{4 1 The} formulation is a well-performing formulation; ^{2 The} formulation is an acceptable ^{formulation 3 The} formulation is a poorly performing formulation ⁴ This formulation is not available.

The currently achieved maximum loading of compound 1A is 2.4%, however, we hope to increase this loading to> 5%.

Generally speaking, PLGA particles include: • Water phase: poly(vinyl alcohol) (PVA), Pluronic F68 (F68), Pluronic F127 (F127), Tween 80. The concentration is given in (w/v)%. • Organic phase: organic solvent, compound 1A and potentially other hydrophobic excipients (such as vanilla extract) Key findings: • NMP is the best solvent for the preparation of PLGA particles. • Pluronic F127 and Tween 80 are some of the best performing surfactants used to prepare the PLGA particles described herein. • Vanilla extract does not increase the drug load of PLGA particles. • Compound 1A is stable during particle generation. • The low concentration of compound 1A in the final suspension is due to its dilution during production. The concentration of compound 1A can be increased by tangential flow filtration.

The LC-UV trace of one of the best performing PLGA formulations for compound 1A is shown in FIG. 6.

Lipid Particles Lipid-based particles as vehicles for compound 1A were studied. The solubility studies of compound 1A in different lipids and oils were performed as shown in the following list. • stearic acid • myristic acid • palmitic acid • isopropyl myristate • isopropyl palmitate • 1-nonanol • linalool • eugenol • trans-cinnamaldehyde • linalyl acetate • p-anisaldehyde • Tetraethylene glycol

Neither the test oil nor the lipid sufficiently dissolved compound 1A . Compound 1A is only soluble in trans-cinnamaldehyde to some extent.

In Example 20, a desolvation method was used, and an organic solvent such as ethanol, acetone or N -methylpyrrolidone was slowly added to the aqueous albumin solution. This will precipitate the albumin. Glutaraldehyde or transglutaminase can be used to crosslink the precipitated protein. The protein particles were purified by centrifugation and resuspended, followed by dialysis.

Example 21 produces albumin nanoparticles by adding urea to the albumin solution. This destabilizes the tertiary structure of the protein, thereby exposing the hydrophobic domain of the protein to the water phase, causing particle formation. Glutaraldehyde or transglutaminase can be used to crosslink the precipitated protein. The protein particles were purified by centrifugation and resuspended, followed by dialysis.

Example 22 is as described in Example 21 but instead destabilizes the protein at high temperature. This also induces a certain amount of protein cross-linking.

Example 23 uses the same procedure as described in Examples 20-22 for this example, except that the protein is silk protein.

Example 24 uses the same procedure as described in Examples 20-22 for this example, except that the protein is gelatin.

Example 25 For any of Examples 20-24, compound 1A was swelled into nanoparticles from a solution in N-methylpyrrolidone or DMSO. Other embodiments

Without departing from the scope and spirit of the present invention, various modifications and changes of the described invention will become obvious to those who are familiar with the art. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed is not limited to these specific embodiments.

Figure 1 shows the challenge with Aspergillus fumigatus AF91 and untreated or with 1.0 mg/kg compound 1A , 0.5 mg/kg compound 1A , 7.5 mg/kg AmBisome, 3.5 mg Chart of survival analysis of mice treated with Ambimide/kg, 7.5 mg/kg voriconazole, 1.0 mg/kg caspofungin or vehicle (5% DMSO containing 5% aqueous glucose). Figure 2 shows the results of the challenge with Aspergillus fumigatus AF91 and untreated or treated with 1.0 mg/kg compound 1A , 0.5 mg/kg compound 1A , 1.0 mg/kg ambiont or 0.5 mg/kg amblyt Graph of mouse survival analysis. Figure 3 shows the challenge with Candida albicans SC5314 without treatment or with 0.7 mg/kg of compound 1A , 0.35 mg/kg of compound 1A , 5.4 mg/kg of Ambidium, 2.7 mg/kg of Ambidium Chart of survival analysis of mice treated with 4 mg/kg voriconazole, 0.35 mg/kg caspofungin, 6 mg/kg fluconazole or vehicle (5% DMSO containing 5% aqueous glucose) . Figure 4 shows mice challenged with Candida albicans SC5314 and untreated or treated with 0.7 mg/kg Compound 1A , 0.35 mg/kg Compound 1A , 0.7 mg/kg Ambibacterium, or 0.35 mg/kg Ambibacterium Chart of survival analysis. Figure 5 is a graph showing the LC-UV trace of Formulation 45 including 1.8% (w/w) Compound 1A. The LC-UV trace exhibited an undesirable reaction between compound 1A and poly(alkyl cyanoacrylate) (PACA) polymer with a residence time of 17-19 min. Figure 6 is a graph showing the LC-UV trace of Formulation 49 including 2.4% (w/w) Compound 1A. The LC-UV trace shows that the degradation or reaction of compound 1A has not occurred during the formulation process. Impurities at 19.5 min are also present in the materials used in the formulation. Figure 7 is a graph showing the LC-UV trace of Formulation 73 including 0.68% (w/w) of Compound 1A. The LC-UV trace shows that the degradation or reaction of compound 1A has not occurred during the formulation process.

Claims (94)

A pharmaceutical composition containing a plurality of nanoparticles, the plurality of nanoparticles containing an active pharmaceutical ingredient, and the active pharmaceutical ingredient is a compound having the following structure:

, Or its pharmaceutically acceptable salt. The pharmaceutical composition of claim 1, wherein the active pharmaceutical ingredient is

, Or its pharmaceutically acceptable salt. The pharmaceutical composition of claim 1 or 2, which further comprises a pharmaceutically acceptable polymer excipient. The pharmaceutical composition of claim 3, wherein the plurality of nanoparticles comprise the pharmaceutically acceptable polymer excipient. The pharmaceutical composition of claim 4, wherein the active pharmaceutical ingredient is nano-encapsulated. The pharmaceutical composition according to any one of claims 3 to 5, wherein the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate) or polyphosphazene. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate). The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable polymer excipient is poly(ethylhexyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(n-hexyl cyanoacrylate) ), poly(4-methylpentyl cyanoacrylate), poly(ethyl butyl cyanoacrylate), poly(butyl cyanoacrylate) or poly(octyl cyanoacrylate). The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable polymer excipient is poly(ethylhexyl cyanoacrylate). The pharmaceutical composition according to any one of claims 3 to 5, wherein the pharmaceutically acceptable polymer excipient is poly(lactic acid-co-glycolic acid). The pharmaceutical composition according to any one of claims 3 to 5, wherein the pharmaceutically acceptable polymer excipient is a protein. The pharmaceutical composition of claim 11, wherein the pharmaceutically acceptable polymer excipient is a protein as casein, albumin, silk protein, gelatin, or a combination thereof. A pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising poly(ethylhexyl cyanoacrylate) and an active pharmaceutical ingredient, the active pharmaceutical ingredient being a compound having the following structure:

, Or its pharmaceutically acceptable salt. A pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising poly(lactic-co-glycolic acid) and an active pharmaceutical ingredient, the active pharmaceutical ingredient being a compound having the following structure:

, Or its pharmaceutically acceptable salt. A pharmaceutical composition comprising a plurality of nanoparticles, the plurality of nanoparticles comprising casein, albumin, silk protein, gelatin or a combination thereof and an active pharmaceutical ingredient, the active pharmaceutical ingredient being a compound having the following structure:

, Or its pharmaceutically acceptable salt. The pharmaceutical composition of claim 12 or 15, wherein the pharmaceutically acceptable polymer excipient is casein. The pharmaceutical composition of claim 12 or 15, wherein the pharmaceutically acceptable polymer excipient is albumin. The pharmaceutical composition of claim 12 or 15, wherein the pharmaceutically acceptable polymer excipient is silk protein. The pharmaceutical composition of claim 12 or 15, wherein the pharmaceutically acceptable polymer excipient is gelatin. The pharmaceutical composition according to any one of claims 1 to 19, wherein the pharmaceutical composition is a lyophilized composition. The pharmaceutical composition according to any one of claims 1 to 19, which further comprises a plurality of microbubbles. A pharmaceutical composition comprising a plurality of microbubbles and a plurality of nanoparticles, the plurality of nanoparticles comprising a compound having the following structure:

, Or its pharmaceutically acceptable salt. The pharmaceutical composition of claim 22, wherein the compound is

, Or its pharmaceutically acceptable salt. The pharmaceutical composition of claim 22 or 23, wherein the nanoparticles comprise poly(alkyl cyanoacrylate), polyphosphazene or poly(lactic-co-glycolic acid). The pharmaceutical composition of claim 22 or 23, wherein the nanoparticles comprise casein, albumin, silk protein, gelatin, or a combination thereof. The pharmaceutical composition according to any one of claims 21 to 25, wherein the microbubbles comprise perfluorocarbon, hydrocarbon, sulfur fluoride gas, air, air component or a mixture thereof. The pharmaceutical composition of claim 26, wherein the microbubbles include nitrogen (N ₂ ), oxygen (O ₂ ), argon (Ar), carbon dioxide (CO ₂ ), helium (He), and neon (Ne) , Methane (CH ₄ ) or a mixture thereof. The pharmaceutical composition of claim 26, wherein the microbubbles comprise perfluorocarbon. The pharmaceutical composition of claim 26, wherein the microbubbles comprise air or its components. The pharmaceutical composition according to any one of claims 21 to 29, wherein at least a part of the plurality of nanoparticles is associated with the surface of the microbubbles. The pharmaceutical composition according to any one of claims 21 to 30, wherein the pharmaceutical composition further comprises a surface active protein. The pharmaceutical composition of claim 31, wherein the surface active protein is albumin. The pharmaceutical composition according to any one of claims 1 to 32, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable surfactant. The pharmaceutical composition of claim 33, wherein the pharmaceutically acceptable surfactant is a nonionic surfactant. The pharmaceutical composition of claim 33, wherein the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitol ester, polysorbate ester, polyethoxylated castor Hemp oil, polyoxyethylene/polyoxypropylene block copolymer or a combination thereof. The pharmaceutical composition of claim 35, wherein the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester or a combination thereof. The pharmaceutical composition of claim 35 or 36, wherein the polyoxyethylene fatty acid ester is polyoxyethylated 12-hydroxystearic acid. The pharmaceutical composition of claim 35 or 36, wherein the polyoxyethylene ether is polyoxyethylene lauryl ether. The pharmaceutical composition according to any one of claims 1 to 38, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable stabilizer. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable stabilizer is vanilla extract, butylated hydroxytoluene, butylated hydroxymethoxybenzene or vitamin E. The pharmaceutical composition of claim 40, wherein the pharmaceutically acceptable stabilizer is vanilla extract. The pharmaceutical composition according to any one of claims 39 to 41, wherein the pharmaceutical composition contains 0.1%-10% (w/w) of a pharmaceutically acceptable stabilizer relative to the mass of the particles. The pharmaceutical composition according to any one of claims 31 to 33, wherein the pharmaceutical composition contains 0.5%-8% (w/w) of a pharmaceutically acceptable stabilizer relative to the mass of the particles. The pharmaceutical composition according to any one of claims 31 to 33, wherein relative to the mass of the particles, the pharmaceutical composition comprises 1%-5% (w/w) of a pharmaceutically acceptable stabilizer. The pharmaceutical composition according to any one of claims 1 to 44, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable oil. The pharmaceutical composition of claim 45, wherein the pharmaceutically acceptable oil is selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, and combinations thereof. The pharmaceutical composition of claim 46, wherein the pharmaceutically acceptable oil is one or more medium chain triglycerides. The pharmaceutical composition of claim 47, wherein the one or more medium-chain triglycerides are selected from the group consisting of Miglyol, Captex, and Kollisolv. The pharmaceutical composition according to any one of claims 45 to 48, wherein relative to the mass of the particles, the pharmaceutical composition comprises 0.5%-5% (w/w) pharmaceutically acceptable oil. The pharmaceutical composition according to any one of claims 1 to 49, wherein the plurality of nanoparticles have an average number average diameter of 20-200 nm as measured by dynamic light scattering. The pharmaceutical composition according to any one of claims 1 to 49, wherein the plurality of nanoparticles have an average number average diameter of 40-100 nm as measured by dynamic light scattering. The pharmaceutical composition according to any one of claims 1 to 49, wherein the plurality of nanoparticles have an average number average diameter of 30-150 nm as measured by nanoparticle tracking analysis. The pharmaceutical composition according to any one of claims 1 to 49, wherein the plurality of nanoparticles have an average number average diameter of 80-100 nm as measured by nanoparticle tracking analysis. The pharmaceutical composition according to any one of claims 1 to 53, wherein the pharmaceutical composition is an aqueous composition. The pharmaceutical composition of claim 54, wherein the pH of the pharmaceutical composition is 4.0 to 8.0. The pharmaceutical composition of claim 55, wherein the pH is 5.0 to 7.0. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition further comprises a co-solvent as a polar organic solvent. The pharmaceutical composition of claim 57, wherein the polar organic solvent is dimethylsulfene, N-methyl-2-pyrrolidone, N,N-dimethylformamide, or a combination thereof. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition contains 1%-15% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition contains 2%-15% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition contains 3%-10% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition contains 3.5%-10% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. The pharmaceutical composition according to any one of claims 1 to 56, wherein the pharmaceutical composition contains 5%-10% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. The pharmaceutical composition according to any one of claims 1 to 63, wherein the pharmaceutical composition contains 3%-6% (dry weight/dry weight) of the active pharmaceutical ingredient as measured by liquid chromatography. A method of treating an individual in need, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 64. The method according to item 65 of the request, wherein the subject has an infection caused by fungi of the following: Candida (Candida), Cryptococcus (Cryptococcus) genus aspergillus (Aspergillus) genus Colletotrichum anthrax bacteria (Colletotrichum) genus, Geotrichum , Hormonema , Lecythophora , Paecilomyces , Penicillium , Rhodotorula , Fusarium ) genus, yeast (Saccharomyces), Trichoderma (Trichoderma) genus Trichophyton (Trichophyton) is a small broom-like mold (Scopularilopsis) species, Histoplasma (Histoplasma) genus, blastomycosis (Blastomyces) or belong to ball Cocciodioides (Cocciodioides) is a genus. The method of claim 66, wherein the individual has a fungal infection caused by: Candida, Aspergillus, or Cryptococcus. The method of claim 67, wherein the individual suffers from a fungal infection caused by azole-resistant Aspergillus sp. The method according to any one of claims 65 to 67, wherein the pharmaceutical composition is intravenously, by inhalation, intranasal, oral, sublingual, buccal, transdermal, intradermal, intramuscular, intravaginal, Parenteral, intraarterial, intracranial, intrathecal, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal or local administration. A method for delivering a therapeutically effective amount of Compound 1 , Compound 1A, or a pharmaceutically acceptable salt thereof to a target site of an individual, the method comprising administering to the individual the pharmaceutical composition according to any one of claims 1 to 64 . The method of claim 70, wherein the pharmaceutical composition is administered intravenously. The method of claim 70 or 71, wherein the target site is the lung of the individual. A method for producing a plurality of nanoparticles, the plurality of nanoparticles comprising a pharmaceutically acceptable polymer excipient and a compound having the following structure:

, Or a pharmaceutically acceptable salt thereof; the method comprises polymerizing a monomer precursor of the pharmaceutically acceptable polymer excipient in a liquid with the compound or a pharmaceutically acceptable salt thereof , The liquid contains the monomer precursor, and the polymerization step produces the plurality of nanoparticles. Such as the method of claim 73, wherein the compound has the following structure:

, Or its pharmaceutically acceptable salt. The method of claim 73 or 74, wherein the liquid further comprises a pharmaceutically acceptable surfactant. The method of claim 75, wherein the pharmaceutically acceptable surfactant is a nonionic surfactant. The method according to any one of claims 73 to 76, wherein the liquid further comprises a pharmaceutically acceptable stabilizer. The method of any one of claims 73 to 77, wherein the liquid further comprises a pharmaceutically acceptable oil. The method of any one of claims 73 to 78, wherein the monomer precursor is alkyl cyanoacrylate, and the pharmaceutically acceptable polymer excipient is poly(alkyl cyanoacrylate). The method according to any one of claims 73 to 79, wherein the plurality of nanoparticles have an average number average diameter of 20-200 nm as measured by dynamic light scattering. The method according to any one of claims 73 to 79, wherein the plurality of nanoparticles have an average number average diameter of 40-100 nm as measured by dynamic light scattering. The method according to any one of claims 73 to 79, wherein the plurality of nanoparticles have an average number average diameter of 30-150 nm as measured by nanoparticle tracking analysis. The method according to any one of claims 73 to 79, wherein the plurality of nanoparticles have an average number average diameter of 80-100 nm as measured by nanoparticle tracking analysis. The method according to any one of claims 73 to 83, wherein the liquid is an aqueous composition. Such as the method of claim 84, wherein the pH of the liquid is 0.5 to 8.0. Such as the method of claim 84, wherein the pH of the liquid is 0.5 to 3.0. Such as the method of claim 84, wherein the pH of the liquid is 2.0 to 8.0. Such as the method of claim 84, wherein the pH of the liquid is 3.0 to 7.0. The method according to any one of claims 73 to 88, wherein the method further comprises adding a plurality of microbubbles. The method according to any one of claims 73 to 89, wherein the method further comprises freeze-drying the plurality of nanoparticles. The method according to any one of claims 73 to 90, wherein the method further comprises dialyzing the plurality of nanoparticles with deionized water. The method according to any one of claims 73 to 91, wherein the method further comprises adjusting the pH of the liquid to a range of 4.0 to 8.0. The method according to any one of claims 73 to 92, wherein the method further comprises adjusting the pH of the liquid to be in the range of 5.0 to 7.0. The method of claim 92 or 93, wherein the step of adjusting the pH is performed during the polymerization step.

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